Post on 03-Apr-2018
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miRCURY LNA Universal RTmicroRNA PCR
Expression
Analysis
Instruction manual v.5.2#203301 March 2013
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Table of contents
Product Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
I. Reagent kits. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
II. Primer Sets and Panels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
Storage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Additional required materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Recommended accompanying products . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
Product description . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
Control Assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
Before starting the Experiment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
Protocols . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
A.Individual assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
B.Human and Mouse&Rat microRNA PCR Panels. . . . . . . . . . . . . . . . . . . . . . . . . . . 29
C.Focus microRNA PCR Panels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36
D.Pick-&-Mix microRNA PCR Panels. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43
Tips to protocol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52
Troubleshooting guide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60
FAQs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61
Related products . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63
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Product summary
The miRCURY LNA Universal RT microRNA PCR system is a microRNA-specific,
LNA-based system designed for sensitive and accurate detection of microRNA by quantitative
real-time PCR using SYBR Green. The method is based on universal reverse transcription
(RT) followed by real-time PCR amplification with LNA enhanced primers (for more details
please see page 12). The miRCURY LNA Universal RT microRNA PCR portfolio is comprised
of four types of reagent kits; including:
Universal cDNA synthesis kit II
RNA Spike-in kit
ExiLENT SYBR
Green master mix kit microRNA primer sets available in pre-defined Human, Mouse&Rat and Focus PCR panels and
customized Pick-&-Mix PCR panels as well as individual primer sets and reference genes. All
PCR panels are Ready-to-Use delivered with one 10 L PCR reaction per well.
Figure 1.
Universal cDNA synthesis kit II
Description page 4
RNA Spike-in kit
(optional)
+
microRNA and referencegene primer sets
Description page 6,protocol page 20
Description page 9,protocol page 43
Description page 7,protocol page 29+36
Pick-&-Mix PCR panels(Ready-to-Use)
+ExiLENT SYBR Green master mix
2,5 or 20ml
Predefined PCR panelsHuman, Mouse&Rat, and Focus PCR panels
(Ready-to-Use)
Description page 5
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I. Reagent kits
Universal cDNA synthesis kit II, 8-64 rxns (product # 203301)
This kit contains all reagents required for first-strand cDNA synthesis for 8-64 reactions1). The
UniSp6 RNA Spike-in template can be used by itself or in combination with the cel-miR-39-3p
template provided in the RNA Spike-in kit:
Contents
5 x Reaction buffer2) 128 L, 5 x concentrated
Enzyme mix 64 L, 10 x concentrated
Nuclease free water 1 ml
UniSp6, RNA Spike-in template 12 fmol, dried down
1) Number of reactions is based on a standard reaction volume of 10 L to 80 L. Reaction volume depends on the application
and number of assays to profile. Please consult Figure 4 for details.2) Includes universal reverse transcr iption primer.3) Used exclusively for the UniSp6 RNA Spike-in control primer set included in the ExiLENT SYBR Green master mix kit.
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ExiLENT SYBR Green master mix, 2.5 ml (product# 203402)
This kit contains all reagents required for PCR amplification of microRNAs. In addition, a
positive control assay, the UniSp6 RNA Spike-in control primer set, is provided with this kit for
amplification of the synthetic UniSp6 RNA spike-in provided in the Universal cDNA synthesis
kit II. The reagents are provided in amounts sufficient for 500 reactions of 10 L.
ExiLENT SYBR Green master mix, 20 ml (product# 203420)
This kit contains all reagents required for PCR amplification of microRNAs. In addition, a
positive control assay, the UniSp6 RNA Spike-in control primer set, is provided with this kit for
amplification of the synthetic UniSp6 RNA spike-in provided in the Universal cDNA synthesis
kit II (the UniSp6 RNA Spike-in control primer set is provided 100x concentration and should
be diluted to 10x concentration before using in the protocol for individual assays). The reagentsare provided in amounts sufficient for 4000 reactions of 10 L.
Contents
PCR Master mix 2 x 1.25 ml, 2x concentrated
Nuclease free water 2 x 1.25 ml
UniSp6 RNA Spike-in control primer set1) 500 L, 10x concentrated
Contents
PCR Master mix 2 x 10 ml, 2x concentrated
Nuclease free water 1 x 20 ml
UniSp6 RNA Spike-in control primer set1) 500 L, 100x concentrated
1) Used exclusively for detection of the UniSp6 RNA spike-in provided with the Universal cDNA synthesis kit II.
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II. Primer Sets and Panels
MicroRNA LNA PCR primer set (product# 204000-205xxx)
LNA PCR primer sets are designed for optimal performance with the Universal cDNA
Synthesis Kit II and the ExiLENT SYBR Green master mix, kit II. The performance of LNA
primer sets will be affected if used in combination with less than optimal reagents. The primer
sets are supplied in sufficient amounts for 200 reactions of 10 L.
Reference gene primer set (product # varies)
The Reference gene primer sets are designed for use with the microRNA primers sets above
as reference genes for normalization. The primer mix is supplied in sufficient amount for 200
reactions of 10 L.
Contents
LNA PCR Primer mix (dried down)
ContentsLNA PCR Primer mix (dried down)
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Pre-defined microRNA PCR Panels (product # varies)
The Human, Mouse&Rat and Focus microRNA PCR panels Ready-to-Use consist of either
96-well or 384-well PCR plates containing dried down LNA primer sets for one 10 L real-
time PCR reaction per well. The LNA primer sets are designed for optimal performance
with the Universal cDNA Synthesis Kit II and the ExiLENT SYBR Green master mix, kit. The
performance of the LNA primer sets will be affected if they are used in combination with
less than optimal reagents.
Contents Human miRNome panel I and II, V3384-well PCR plates supplied with LNA primer sets dried down, one 10 L reaction per well:
Contents Mouse&Rat miRNome panel I and II, V3
384-well PCR plates supplied with LNA primer sets dried down, one 10 L reaction per well:
Panel I Panel II
372 LNA primer sets for theamplification of human microRNAs1)
380 LNA primer sets for theamplification of human microRNAs1)
3 inter-plate calibrators 3 inter-plate calibrators
3 primer sets for reference genes2) 1 blank well
5 RNA Spike-in control primer sets 3)
1 blank well
Panel I Panel II
372 LNA primer sets for the
amplification of mouse and rat microRNAs1)
380 LNA primer sets for the
amplification of mouse and rat microRNAs1)
3 inter-plate calibrators 3 inter-plate calibrators
3 primer sets for reference genes2) 1 blank well
5 RNA Spike-in control primer sets 3)
1 blank well
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Contents Serum/Plasma Focus microRNA PCR panel, V3
PCR plates compatible with various real-time PCR instruments are available and supplied
with LNA primer sets dried down, one 10 L reaction per well:
Contents Cancer Focus microRNA PCR panel, V3
PCR plates compatible with various real-time PCR instruments are available and supplied
with LNA primer sets dried down, one 10 L reaction per well:
96-well (2 plates) 384-well plate (2 panels per plate)
179 LNA primer sets for the amplificationof human microRNAs1+2)
2x179 LNA primer sets for the amplification of humanmicroRNAs1+2)
2x3 Inter-plate calibrators 2x6 Inter-plate calibrators
5 RNA Spike-in control primer sets 3) 2x5 RNA Spike-in control primer sets3)
2 blank wells - 1 in each plate 2x2 blank wells
96-well (1 plates) 384-well plate (4 panels per plate)
84 LNA primer sets for the amplificationof human microRNAs1)
4x84 LNA primer sets for the amplification of humanmicroRNAs1)
3 Primer sets for potentialreference genes2)
4x3 Primer sets for potentialreference genes2)
3 Inter-plate calibrators 4x3 Inter-plate calibrators
5 RNA Spike-in control primer sets 3) 4x5 RNA Spike-in control primer sets 3)
1 blank well 1 blank well
1) Please go to www.exiqon.com/mirna-pcr to download plate layout files.2) Human Panels and Cancer Focus Panel: Three snRNAs (U6snRNA, SNORD38B, SNORD49A). Mouse&Rat Panels: Three
snRNAs (U6snRNA, RNU5G, RNU1A1). Serum/plasma Focus Panel: miR-103, miR-191, miR-423-5p, miR-16, miR-425,
miR-93, miR-451 are regarded reference gene candidates.3) The RNA Spike-in control primer sets targets the UniSp6 RNA spike-in supplied in the Universal cDNA synthesis kit II
and the 4 RNA spike-ins contained in the RNA Spike-in kit (UniSp2, UniSp4, UniSp5, and cel-miR-39-3p).
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Pick-&-Mix microRNA PCR Panel, (product # 203801 and 203802)
The Pick-&-Mix microRNA PCR Panels consist of either 96-well PCR plates or 384-well
PCR plates containing custom selections of dried down microRNA LNA PCR primer sets
for one 10 L real-time PCR reaction per well, ready-to-use. PCR plates compatible with
various real-time PCR instruments are available. The LNA primer sets are designed for
optimal performance with the Universal cDNA Synthesis Kit II and the ExiLENT SYBR Green
master mix kit. The performance of the LNA primer sets will be affected if they are used in
combination with less than optimal reagents.
Contents
PCR plates supplied with customer defined LNA primer sets, reference gene primer sets,
and RNA Spike-in control primer sets, dried down, one 10 L reaction per well1):
96-well PCR plates1) 384-well PCR plates1)
10 primer sets in 8 replicates 22 primer sets in 16 replicates
22 primer sets in 4 replicates 46 primer sets in 8 replicates
92 primer sets in 1 replicate 94 primer sets in 4 replicates
96-well flexible layout 380 primer sets in 1 replicate
384-well flexible layout
1) Please go to www.exiqon.com/pick-and-mix to configure a Pick-&-Mix microRNA PCR Panel.
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Storage
All microRNA PCR Panels, LNA primer sets and Reference gene primer sets
The PCR panels and primer sets are shipped dried down at room temperature. The primers
can be stored between +4C and -20C. Under these conditions, all components are stable
for at least 12 months. After resuspension, it is recommended to store LNA primer sets
and Reference gene primer sets in aliquots at -20C to avoid repeated freeze-thaw cycles.
Universal cDNA synthesis kit II and ExiLENT SYBR
Green master mixThese kits are shipped on dry ice in polystyrene containers and should be stored at -15C to
-25C. Do not store in a frost-free freezer. Under these conditions, all components are stable
until the expiry date on the package or vial. It is recommended that the RNA spike-in be stored
in aliquots at -20C after re-suspension to avoid repeated freeze-thaw cycles.
Additional required materials
Reagents not supplied
ROX or other passive reference dye (required on some PCR cyclers)
Materials and Equipment not supplied
Nuclease-free PCR tubes or plates for use with individual assays
Nuclease-free, aerosol barrier pipette tips
Nuclease-free, low nucleic acid binding microcentrifuge tubes
(e.g. Eppendorf DNA LoBind tubes product and original NUNC vials)
Sealing foils for PCR plates
Micro-centrifuge and plate centrifuge
Heating block, thermal cycler or other incubators
Real-time PCR instrument
Optional but recommended product
miRCURY LNA Universal RT microRNA PCR, RNA Spike-in kit
(product# 203203)
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Recommended accompanying products
Exiqon recommends the Exiqon GenEx qPCR software for comprehensive and convenient data
analysis. GenEx includes a wizard for import of Exiqon miRCURY Universal RT microRNA PCR
data and offers advanced methods to analyze real-time qPCR data in a few simple steps. The
software includes tools for selection and validation of reference genes, data pre-processing and
comprehensive statistical analyses. For more information and to download a free trial, please
go to www.exiqon.com/qpcr-software. The following Exiqon GenEx products are available:
Exiqon GenEx Industrial - Exiqon version of GenEx,
qPCR analysis software, industrial license
Exiqon GenEx Academic - Exiqon version of GenEx,
qPCR analysis software, academic license
Exiqon recommends the miRCURY RNA Isolation kits for purification of total RNA or small
RNA fraction. RNA purified using the miRCURY RNA Isolation kits is fully compatible with
the miRCURY LNA Universal RT microRNA PCR System. The following kits are available:
miRCURY RNA Isolation Kit Cell & Plant
Provides a rapid method for purification of total RNA from cultured animal cells, small tissue
samples, blood, bacteria, yeast, fungi and plants.
miRCURY RNA Isolation Kit Tissue
Specifically designed for purification of total RNA from tissue samples.
miRCURY RNA Isolation Kit Biofluids
Kit for purification of low abundant small RNAs from samples such as serum, plasma, urine
and CSF.
See Tip 2
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Product description
A unique system for microRNA profiling
miRCURY LNA Universal RT microRNA PCR offers the best available combination of
performance and ease-of-use on the microRNA real-time PCR market because it unites two
important features (Figure 2):
1. Universal RT One first-strand cDNA synthesis reaction provides template for all microRNA
real-time PCR assays. This saves precious sample, reduces technical variation, requires use
of less reagents, and saves time in the laboratory.
2. LNA PCR amplification Both PCR amplification primers (forward and reverse) are
microRNA specific and optimized with LNA. The result is: 1) exceptional sensitivity as well as
extremely low background enabling accurate quantification of very low microRNA levels and 2)
highly specific assays that allow discrimination between closely related microRNA sequences.
miRCURY LNA Universal RT microRNA PCR offers solutions both for high-throughput
microRNA expression profiling and for quantification of individual microRNAs.
Step 1: First-strand synthesis (RT)
Step 2: Real-time PCR amplification
Mature microRNAAAAAAAAAAAAAAAAAAAAAA)
3 degenerate anchor
miR-specific forward primer
miR-specific reverse primer
AAAAAAAAAAAAAAAAAAAATTTTTTTTTTTTTTT
TTTTTTTTTTTTTTT
5 universal tag
B)
A)
B)
Figure 2. Schematic outline of the miRCURY LNA Universal RT microRNA PCR System. A poly-A tail is added to the
mature microRNA template (step 1A). cDNA is synthesized using a poly-T primer with a 3 degenerate anchor and a 5
universal tag (step 1B). The cDNA template is then amplified using microRNA-specific and LNA-enhanced forward and
reverse primers (step 2A). SYBR Green is used for detection (step 2B).
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Control Assays
There are 3 different types of control assays available in the miRCURY LNA Universal RT
microRNA PCR system:
Reference assays and reference candidates
Inter-plate calibrators
RNA spike-in assays
All of these control assays are available in the microRNA PCR panels. One RNA spike-in templateis provided with the Universal cDNA Synthesis Kit II, while the assay that will detect this RNA
spike-in is available in the ExiLENT SYBR Green master mix. Additionally 4 RNA spike-in
templates are available as a spike-in kit. The assays for detection of these 4 templates as well
as the reference assays are available as individual primer sets.
Reference assays and reference candidates
These assays detect small non-coding RNAs - either small nuclear RNA, small nucleolar RNA or
microRNA which frequently are found to be stably expressed across different cells or tissues.
Reference assays may therefore be candidate assays for normalization in a profiling study withseveral samples. Though this is a good and recommended approach, great caution should be taken
in the selection of reference genes. The danger of using endogenous reference genes lies in the
assumption that a specific gene is expressed at the exact same level in all sample types. This is
rarely true. The selection of reference genes should therefore be made with care, and should be
specific to the sample set you are working with. The actual selection of reference genes to be used for
normalization should always be based on a determination of the most stably expressed gene(s) which
may be done using either GeNorm or NormFinder both tools that are integrated within Exiqons
GenEx data analysis software. When applicable, we recommend using microRNA rather than small
nuclear RNA or small nucleolar RNA for normalization. Firstly, small nuclear and nucleolar RNAs
are longer RNA species than microRNA and may purify differently from microRNA. Moreover, small
nuclear and nucleolar RNAs have entirely different functions as well as sub-cellular locations, and
finally certain samples like blood plasma does not contain the small nuclear and nucleolar RNAs.
Global mean normalization is a preferred alternative to using reference genes for normalization
when working with panels and samples where many microRNAs are screened per sample and where
many microRNA are called (detected) in all samples. Exiqons GenEx will easily perform global mean
normalization. To read more on normalization.
See Tip 11
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Inter-plate calibrators
Three wells within the pre-defined Human, Mouse&Rat, and Focus PCR Panels contain the
inter-plate calibrator assay (annotated as UniSp3 IPC in the plate layout files). Depending
on the plate layout, the Pick-&-Mix Panels contain at least three inter-plate calibrators.
Each of these wells, contain a pre-aliquoted primer pair and a DNA template and therefore
the variation is very minimal from well-to-well and from plate-to-plate of these assays. The
inter-plate calibrators are used for calibration between PCR plate runs which is very useful
on some instruments that apply the cycle threshold method for Cq determination such as
the ABI7900 PCR cycler. Since the inter-plate calibrators are independent of cDNA quality inorder to give a signal (but may be affected by PCR inhibitors in the sample) they may be used
to quality control each plate run.
Inter-plate calibration (IPC) can easily be performed in the data analysis software Exiqons
GenEx. Alternatively, IPC may be performed manually by using the IPC assay replicates as
follows. For each plate, verify that the replicates have Cq standard deviation within 0.5. If
this is not the case, eliminate the outlier if this can be identified. Calculate the average of
the replicates for each plate, the overall average (average of IPC values from all plates). The
calibration factor is calculated as the difference between plate average and overall averagefor each plate (calibration factor = IPCplate-IPCoverall). An example is shown in Table 1.
Finally, calibrate each plate by subtracting the calibration factor from all Cq values in the plate.
Table 1. Example of inter-plate calibration (IPC)
Plate 1 Plate 2 Plate 3
let-7c 21.12 20.93 21.34
IPC plate average 19.72 19.70 20.00
IPC overall average 19.81 19.81 19.81
Calibration factor -0.09 -0.11 0.19
let-7c calibrated 21.21 21.04 21.15
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RNA spike-ins (synthetic control templates)
The primary purpose of the RNA spike-ins and the matching primer pairs for detection of
these is to provide controls for the quality of the RNA isolation, the cDNA synthesis reaction
and the PCR. RNA isolations may vary in yield, purity and integrity. Some sample types may
contain compounds that inhibit the cDNA synthesis or the PCR even though the RNA has been
purified using the best standard procedures. This may result in different efficiencies of the
reverse transcription or PCR between compared samples. One way to control for differences
in efficiencies at each experimental level (isolation, cDNA synthesis, and PCR) is by adding
known RNA spike-ins to the sample prior to isolation and cDNA synthesis. Use of the RNAspike-ins may also reveal if nucleases are present. After conducting the PCR but before
initiating the data analysis, wells detecting RNA spike-ins are compared and outlier samples
may be identified and considered for exclusion in the further data analysis.
We have designed a flight of RNA spike-ins for this purpose. The UniSp6 RNA Spike-in
template is provided with the Universal cDNA synthesis kit II. Additionally four RNA spike-
in templates can be obtained with the separate RNA Spike-in kit. Here, a vial of three RNA
spike-in templates, UniSp2, UniSp4 and UniSp5, mixed at different concentrations can be
used during RNA isolation. The cel-miR-39-3p RNA template provided in a separate vial in theRNA Spike-in kit can be mixed with the UniSp6 template from the Universal cDNA synthesis
kit II to obtain two different template concentrations. This combination can be added during
the cDNA synthesis. Five wells in pre-defined PCR panel plates contain the matching primer
sets. The selection of RNA Spike-in control primer set in the Pick-&-Mix PCR Panel can be
customized to the specific need. A UniSp6 control primer set is also provided with the ExiLENT
SYBR Green master mix kit, which is to be used with our non-plate based PCR primer set
products.The RNA spike-ins are shipped dried down and must be re-suspended before use.
If the RNA Spike-in kit with multiple RNA spike-ins is used, follow the protocol accompanying
that product.
If UniSp6 is to be used alone:
1. Re-suspend the UniSp6 RNA spike-in by adding 80 L nuclease free water to the tube.
2. Mix by vortexing and spin down. Leave for 20-30 min. on ice to fully dissolve RNA spike-in.
Mix by vortexing and spin down. Store in aliquots at -20C
3. Prior to the RT reaction, add 1 L synthetic spike-in (108 copies/L) per 20 ng sample RNA.
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An alternative application of the UniSp6 RNA spike-in is as inter-plate calibrator. This
is only relevant when using individual LNA primer sets and Reference gene primer
sets in a multi-plate set-up. The microRNA PCR panels already contain an inter-plate
calibrator. Add 1 L synthetic spike-in (108 copies/L) to 20 ng of a complex RNA sample
(e.g. total RNA from MS2, yeast, or a cell line; not provided with the kit). Proceed with first
strand synthesis and subsequently real-time PCR as described in the protocols of the current
instruction manual. At least one spike-in amplification reaction per PCR plate is used for
inter-plate calibration.
Experimental design
Before starting the experiment, it is essential to consider the experimental setup and consider
the number of replicates needed for obtaining significant results replicates being technical
as well as biological. The number of biological replicates required varies from experiment to
experiment depending on the variation within and between the groups. We recommend that a
No Template Control (NTC) is included in the study every time a new experiment is set up, to
set the background level. The most optimal NTC is a mock up sample preparation including
only carrier RNA as sample. The NTC should be run on all assays included in the study. Wedefine the background of an assay as 5 Cq values below the NTC level. Furthermore we
recommend including spike-ins found in the Spike-in kit to provide full quality control over
all steps in the profiling (see figure 3). Finally it is necessary to include a number of candidate
reference miRNAs, which are expected to be constitutively expressed across the different
experimental conditions, for data normalization.
Figure 3. Experimental design
See Tip 9
UniSp2
Sample n
Sample n+1 Sample preparation evaluation
Sold as separate Spike-in kit (203203)
cDNA synthesis evaluation
Comes withUniversal cDNAsynthesis kit II
Included in allpanel products
Assays available asindividual assays or inready-to-use panels
Inter plateCalibration
Sample profiling
NTC
UniSp4 UniSp5 cel-miR-39-3p UniSp6 UniSp3 miR-A miR-X
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Before starting the Experiment
Before setting up a real-time PCR experiment, there are a number of practical experimental
design parameters that should be considered:
RNA input - The miRCURY LNA Universal RT microRNA PCR protocol is optimized for use
of 20 ng total RNA per 20L cDNA synthesis reaction. The exact amount of total RNA needed
depends on whether the downstream application is individual assays or panels. Furthermore,
the amount of total RNA to be used may also vary depending on the microRNA expression
levels in the cells or tissue to be analyzed. For highly expressed microRNAs it is possible to
use down to 10 pg total RNA as starting material. For weakly expressed microRNAs it may
be possible to use up to 200 ng of total RNA; however, in samples with high amounts of PCR
inhibitors (e.g. FFPE tissue samples), this may not be feasible. Finally, inhibitors may be presentin RNA preparations from certain samples e.g. serum and plasma. Prior to conducting a
larger microRNA profiling study, it is recommended to optimize the amount of input RNA to
the RT reaction in order to avoid conducting a larger study where inhibition occurs sporadically
throughout the data set.
Information on how to extract and handle RNA can be found in the tips section. In short, total
RNA should be prepared using a method that preserves small RNA species. DNase treatment
may be necessary. When using commercially available kits, please ensure that the total RNA
preparation is guaranteed to contain microRNAs.
RNA work requires specific handling and precautions to prevent RNase contamination of
the reagents and degradation of the RNA sample. Find information on how to handle RNA
in the tips section starting on page 52. The tips section also provides simple guidelines
for good laboratory practice to ensure optimal performance of PCR experiments.
Important note
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Note: Blood serum and plasma are particular sample types that require special RNA purification
procedures and the amount of RNA present in the samples can usually not be accurately
determined. Due to the low levels of microRNAs and potentially high levels of inhibitors
in samples derived from serum and plasma, specific recommendations for how to set up
experiments using these types of sample can be found in the miRCURY LNA Universal RT
microRNA PCR, Instruction Manual Biofluid samples.
Normalization when running individual assays or when configuring a Pick-&-Mix microRNA PCR
panel it is important to consider how the data will be normalized. For tips and recommendationson choosing the correct reference genes and how to test and validate reference genes, please
see section on reference assays (page13) and the Tip 11 (page 58).
Excess volumes required for pipetting Liquid handling with pipettes or pipetting robots require
excess volumes of reagents due to loss during pipetting. The loss depends on the available
pipetting system but losses in the range from 10% -25% are not uncommon. All protocols in the
current instruction manual reflect the required reaction volumes and pipetting volumes should
be adjusted according to accommodate the pipetting loss of the available pipetting system.
ROX ROX is a passive reference dye used by some PCR cycles to obtain a robust read over
the entire array of wells in a 96- or 384-well PCR plate. The requirement for ROX is instrument
dependent and we recommend to follow the instrument manufactures guidelines on this (see
also Tip 8).
ABI instruments the default settings on ABI real-time PCR cyclers are not suitable for
running miRCURY LNA Universal RT microRNA PCR. Settings need to be changed from
automatic to manual background and threshold settings to obtain valid PCR data (see also Tip
10). Furthermore, if the dataset is to be analyzed using the GenEx software, it is important that
the experiment is set up as an AQ experiment, not RQ. To ensure correct settings, download
the instrument settings file at www.exiqon.com/sds.
See Tip 8
See Tip 10
See Tip 11
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RNA spike-ins consider how the RNA spike-ins should be applied in the planned study.
Please consult the section on RNA spike-ins on page 15.
Protocols for the first-strand cDNA synthesis and real-time amplication follows on the
next pages:
A. Individual assays, please go to page 20
B. Human and Mouse&Rat microRNA PCR Panels, please go to page 29
C. Focus microRNA PCR Panels, please go to page 36
D. Pick-&-Mix microRNA PCR Panels, please go to page 43
Figure 4 gives an overview and helps identifying which protocol to follow as well as the
recommended cDNA reaction volume needed for a given sample and assay type.
Figure 4. Protocol overview
Cells or tissue Serum plasma or other biofluidsSample type
Panels orprimersets:
UniversalcDNAreactions perkit
UniversalcDNAreactionvolume
miRNomepanel I
miRNomepanel I+II
1-96 97-192
Individualassays(
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Protocol A - Individual assays
This protocol is used for conducting the first-strand cDNA synthesis and real-time PCR, using
the individual assays for human, mouse and rat (product numbers 204000-205xxx).
If working with serum plasma samples or other biofluids, please refer to the specific miRCURY
LNA Universal RT microRNA PCR Instruction Manual for biofluid samples at
www.exiqon.com/serum-plasma-pcr-manual.
Before using the LNA PCR primer mix or the Reference gene primer mix for the first time,
the primers must be re-suspended: Re-suspend the primer mix by adding 220 L nuclease free water to the tube. Mix by
vortexing and spin down. Leave on ice for 20-30 minutes.
The UniSp6 RNA spike-in control primer mix is supplied with the master mix and is already
dissolved. Ensure primer concentration is 10x before proceeding with the protocol (see
page 5 for details).
Additional required materials:
96- or 384-well plate real-time PCR cycler Thermocycler for first-strand cDNA synthesis
96/384-well plates or tube strips compatible with available real-time PCR cycler
Micro centrifuge
Swing bucket centrifuge for 96-/384-well plates
Protocol-
Individualassays
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Checklist:
Have you considered excess volumes required for using liquid handling robotics see page 18
Did you consider how to use the RNA spike-ins please see page 15
ROX: The ExiLENT SYBR Green master mix, does not include the ROX passive reference
dye. Please follow instrument manufactures recommendations
ABI instruments: The use of manual background and threshold settings is necessary for
obtaining correct PCR data. Make sure to have the optimal settings by downloading the
instrument settings file at www.exiqon.com/sds. Furthermore, if the data is to be analyzed
using GenEx, the experiment must be set up as an AQ experiment, not RQ Have you optimized the input amount to the RT reaction in order to avoid inhibition?
Protocol-
In
dividualassays
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Phase I:Prepare RNA sampleSee page 52 for recommendations
Phase II: cDNA synthesisSee protocol page 23.
Phase III: real-time PCR amplificationSee protocol page 25.- Prepare adequate amount of pre-mixed primer+ PCR Master mix and distribute into wells
- Add cDNA to all primer sets to be analyzed
Phase IV: Data analysisSee data analysis guide online- Export data for further analysis- Data pre-processing, normalization
and statistical analysis
4
3
2
1
0
-1Normal Tumor Tumor
stroma
Tumor Total
miR-21
let-7a
Relativeexpression
(log2)
LNATM
primer sets
Workflow for individual primer sets (per sample)
Protocol-
Individualassays
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Protocol
The miRCURY LNA Universal RT microRNA PCR protocol is a two-part protocol consisting of:
1. First-strand cDNA synthesis (Step 1-5)
2. Real-time PCR amplification (Step 6-11)
Important: Keep reagents and reactions on ice (or at 4C) at all times.
First strand synthesis
Step 1
Dilute template RNA
Adjust each of the template RNA samples to a concentration of 5 ng/L
using nuclease free water.
Step 2
Prepare reagents
Gently thaw the 5x Reaction buffer and nuclease-free water, and
immediately place on ice. Mix by vortexing. Re-suspend the RNA spike-
ins according to the appropriate RNA Spike-ins protocol (see page 15),
leave on ice for 15-20 minutes. Immediately before use, remove the
Enzyme mix from the freezer, mix by flicking the tubes and place on
ice. Spin down all reagents.
Protocol-
In
dividualassays
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Step 4
Mix and spin
reagents
Mix the reaction by very gentle vortexing or pipetting to ensure that all
reagents are thoroughly mixed. After mixing, spin down.
Step 5
Incubate and heat
inactivate1)
Incubate for 60 min at 42C.
Heat-inactivate the reverse transcriptase
for 5 min at 95C. Immediately cool to 4C.
Store at 4C or freeze.
Step 3
Combine reagents
according to Table 2
Note: remember to
calculate necessary
excess volume for
pipetting and robotic
dead volume.
If performing first-strand cDNA synthesis on multiple RNA samples, it
is recommended to prepare an RT working solution of the 5x Reaction
buffer, water, Enzyme mix and RNA spike ins (in the proportion indicated
in the first four lines of Table 2).
The following procedure is recommended:
1. Prepare the required amount of RT working solution and place it on ice.
2. Dispense RT working solution into nuclease free tubes.
3. Dispense template RNA in each tube.
Table 2 Reverse transcription reaction setup
Reagent Volume (L),RT reaction
5x Reaction buffer 2
Nuclease-free water 4.5
Enzyme mix 1
Synthetic RNA spike ins, optionalreplace with H
2O if omitted
0.5
Template total RNA (5 ng/L) 2
Total volume 10
1) The protocol can be interrupted at this stage. The undiluted cDNA may be kept at -20C for up to 5 weeks (optional store
at 4C for up to 4 days). It is recommended that synthesized cDNA is stored in low-nucleic acid binding tubes or plates.
Protocol-
Individualassays
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qPCR protocol
Step 6
Prepare reagents for
real-time PCR
Place cDNA (from Step 5), nuclease free water and PCR Master mix on
ice and thaw for 15-20 min. Protect the PCR Master mix vials from light.
Immediately before use, mix the PCR Master mix by pipetting up and
down. The rest of the reagents are mixed by vortexing and spun down.
Step 7
Dilute cDNA template
80x in nuclease
free water2)
Immediately before use, dilute only the amount of cDNA template
needed for the planned real-time PCR reactions 80x in nuclease free
water (e.g. add 395 L nuclease free water to each 5 L of reaction). It
is important that low-nucleic acid binding tubes or plates are used. It
is not recommended to store the 1:80 dilution of cDNA.
Recommendation: Include a passive reference dye in the cDNA
dilution if advised by instrument manufacturer. Please note that
the PCR Master mix does not include ROX. The amount of ROX
required is instrument dependent and it is important to refer to the
manufacturers recommendations when deciding how much ROX
to use, see Tip 8.
2) Adjust volumes to accommodate your in-house liquid handling system volume loss when pipetting
See Tip 8
Protocol-
In
dividualassays
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Step 9
Mix and spin
reagents
Mix the reaction by gentle pipetting to ensure that all reagents are mixed
thoroughly. After mixing cap tubes or strips or seal the plate with optical
sealing as recommended by the manufacturer. Spin down in a centrifuge
(1500g for 1 minute). The experiment can be paused at this point. Store
the reactions protected from light at 4C for up to 24 hours.
Step 8
Combine PCR Master
mix, PCR primer mix
and cDNA according
to Table 3.
Note: remember to
calculate necessary
excess volume for
pipetting and robotic
dead volume.
When multiple real-time PCR reactions are performed with the same
microRNA primer set, it is recommended to prepare a primer master
mix working-solution of the PCR primers and the PCR Master mix (in
the proportion indicated in Table 3).
The following procedure is recommended:
1. Prepare the required amount of primer:master mix working-solution
(see Table 3) and place it on ice. It is recommended to include excess
of all reagents in the master mix to compensate for pipetting excess
material.
2. Place the relevant volume of primer:master mix working-solution
in PCR tubes/wells (see Table 3) and spin tubes/plate briefly in a
centrifuge (1500g for 1 minute), to remove air bubbles.
3. Add cDNA template to each tube/well.
Table 3 Real-time PCR reaction, pr. 10 L reaction3)
Reagent Volume (L), 96/384-well plate,tubes or strips
PCR Master mix 5
PCR primer mix4) 1
Diluted cDNA template 4Total volume 10
3) If using a 96-well cycler with a minimum recommended volume of 20 L (as some ABI instruments), then use 10 L
reaction volume and set the instrument settings at 20 L.4) The PCR primer mix must be dissolved prior to real-time PCR set-up, see page 20.
Protocol-
Individualassays
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Step 10
Real-time PCR
amplification
Perform real-time PCR amplification followed by melting curve analysis
according to Table 4.
Table 4 Real-time PCR cycle conditions
Process step Settings, LC480instrument5)
Settings, otherinstruments3)
Polymerase Acti vation/Denaturation 95C, 10 min 95C, 10 min
Amplification 45 amplificationcycles at95C, 10 s60C, 1 min,ramp-rate1.6C/s6)
Optical read
40 amplificationcycles at95C, 10 s60C, 1 min,ramp-rate1.6C/s6)
Optical read
Melting curve analysis7) Yes Yes
Step 11
Analyze data
Perform initial data analysis using the software supplied with the real-
time PCR instrument to obtain raw Cq values (Cp or Ct, depending on
PCR instrument). If you are using an ABI instrument, please note that
it is not recommended to use auto Ct settings. Furthermore, if the data
is to be analyzed using Exiqon GenEx, the experiment must be set up as
an AQ experiment, not RQ. For a guide on how to set manual baseline
and threshold, refer to Tip 10, page 56 in the tips section. If you are
using a Roche LC480 instrument, we recommend analysis using the
2nd derivative method.
For tips on normalization, please see Tip 11, page 58. We
recommend performing normalization and further data analysis
with the Exiqon GenEx qPCR analysis software (www.exiqon.
com/mirna-pcr-analysis). Please refer to our data analysis guide
for recommendations.
ABI instrument user?
Apply manual baseline and threshold settings! Go to www.exiqon.com/sds to download settings file
See Tip 11
Protocol-
In
dividualassays
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5) Five additional amplification cycles are required when using the LC480 instrument to allow collection of assay datawith Cp-values up to 40.
6) The ramp-rate of cooling from 95C to 60C should be set to 1.6C/s. This is equivalent to 100% under standard cycling
conditions on the ABI 7500, 7900 and Viia7 instruments. If the ramp rate of cooling is too rapid, performance may be
compromised.7) Melting curve analysis of the PCR product(s) is recommended to verify specificity and identity of the amplification reaction.
Melting curve analysis is an analysis step built into the software of instruments. Please follow the instructions provided by
the supplier. Note: The Tm of a PCR product depends on buf fer composition, salt concentration and the PCR instrument.
Protocol-
Individualassays
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Protocol B - Human and Mouse&RatmicroRNA PCR Panels
This protocol is used for conducting the first-strand cDNA synthesis and real-time PCR using
the following products:
Human miRNome PCR Panel (product numbers 203611 to 203614)
Mouse&Rat miRNome PCR Panel (product numbers 203709 to 203712)
If working with serum plasma samples or other biofluids, please refer to the specific
miRCURY LNA Universal RT microRNA PCR Instruction Manual for biofluid samples at
www.exiqon.com/serum-plasma-pcr-manual.
Additional required materials:
384-well plate real-time PCR cycler
Thermocycler for first-strand cDNA synthesis
Micro centrifuge
Tube for mixing water and master mix (10 ml)
Sealing foils for PCR plates
Swing bucket centrifuge for 96/384-well plates
Recommended: Liquid handling robot for pipetting
Checklist:
Have you considered excess volumes required for using liquid handling
robotics see page 18
Did you consider how to use the RNA spike-ins please see page 15
ROX: The ExiLENT SYBR Green master mix, does not include the ROX passive reference
dye. Please follow instrument manufactures recommendations
ABI instruments: The use of manual background and threshold settings is necessary
for obtaining correct PCR data. Make sure to have the optimal settings by downloading
the instrument settings file at www.exiqon.com/sds. Furthermore, if the data is to be
analyzed using GenEx, the experiment must be set up as an AQ experiment, not RQ.
Have you optimized the input amount to the RT reaction in order to avoid inhibition?
Protocol-Human,
Mo
use&RatPanels
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Workflow for Human and Mouse&Rat microRNA PCR Panels (per sample)
Protocol-Hum
an,
Mouse&RatPanels
Phase I:Prepare RNA sampleSee page 52 for recommendations
Phase II: cDNA synthesisSee protocol page 31.
Phase III: real-time PCR amplificationSee protocol page 33.- Mix cDNAs with PCR Master mix- Add cDNA:PCR Master mix to
PCR plates
Phase IV: Data analysisSee data analysis guide online- Export data for further analysis- Data pre-processing, normalization
and statistical analysis
4
3
2
1
0
-1Normal Tumor Tumor
stroma
TumorT otal
miR-21let-7a
Relativeexpression
(log2)
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P r
ot oc
ol
-H
um
an,
M o
use&
R a
tP
an
el s
Protocol
The miRCURY LNA Universal RT microRNA PCR protocol is a two-part protocol consisting of:
1. First-strand cDNA synthesis (Step 1-5)
2. Real-time PCR amplification (Step 6-9)
Important: Keep reagents and reactions on ice (or at 4C) at all times.
First strand synthesis:
Step 1
Dilute template RNA
Adjust each of the template RNA samples to a concentration of 5 ng/l
using nuclease free water.
Step 2
Prepare reagents
Gently thaw the 5x Reaction buffer and nuclease-free water, and
immediately place on ice. Mix by vortexing. Re-suspend the RNA spike-
in(s) according to the appropriate RNA Spike-in protocol (see page 15),
leave on ice for 15-20 minutes. Immediately before use, remove the
Enzyme mix from the freezer, mix by flicking the tubes and place on
ice. Spin down all reagents.
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miRCURY LNA Universal RT microRNA PCR Instruction Manual
Protocol-Hum
an,
Mouse&RatPanels
Step 4
Mix and spin
reagents
Mix the reaction by very gentle vortexing or pipetting to ensure that all
reagents are thoroughly mixed. After mixing, spin down.
Step 5
Incubate and heat
inactivate1)
Incubate for 60 min at 42C.
Heat-inactivate the reverse transcriptase
for 5 min at 95C.
Immediately cool to 4C.
Store at 4C or freeze.
Step 3
Combine reagents
according to Table 5
Note: remember to
calculate necessary
excess volume for
pipetting and robotic
dead volume.
If performing first-strand cDNA synthesis on multiple RNA samples, it
is recommended to prepare an RT working solution of the 5x Reaction
buffer, water, Enzyme mix and RNA spike ins (in the proportions indicated
in the first four lines of Table 5).
The following procedure is recommended:
1. Prepare the required amount of RT working solution and place it on ice.
2. Dispense RT working solution into nuclease free tubes.
3. Dispense template RNA in each tube.
Table 5 Reverse transcription reaction setup
1) The protocol can be interrupted at this stage. The undiluted cDNA may be kept at -20C for up to 5 weeks (optional store
at 4C for up to 4 days). It is recommended that synthesized cDNA is stored in low-nucleic acid binding tubes or plates.
Reagent Panel IVolume (L)
Panel I+IIVolume (L)
5x Reaction buffer 4 8
Nuclease-free water 9 18
Enzyme mix 2 4
Synthetic RNA spike ins, optionalreplace with H2O if omitted
1 2
Template total RNA (5ng/L) 4 8
Total volume 20 40
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Protocol-Human,
Mo
use&RatPanels
qPCR protocol:
Step 7
Combine PCR Master
mix, water and cDNA
and add to PCR plates2)
The following procedure is recommended to avoid low concentrations
of cDNA from adhering to tube surface:
1. Before removing the plate seal, briefly spin
down the plate(s) in a plate centrifuge.
2. Combine 2x PCR Master mix and water. Panel I: 2000 L 2x master
mix and 1980 L water, Panel I+II: 4000 L 2x master mix and 3960
L water.
3. Mix gently and spin down.
4. Add 20 L cDNA (panel I) or 40 L cDNA (panel I+II) and mix.
5. Add 10 L PCR Master mix: cDNA
mix to each well3).
6. Seal the plate with optical sealing as recommended by theinstrument manufacturer.
7. Spin plate briefly in a plate centrifuge (1500g for
1 minute), to to collect the sample.
The experiment can be paused at this point. Store the reactions protected
from light at 4C for up to 24 hours.
Recommendation: Include a passive reference dye in the cDNA dilution if
advised by instrument manufacturer. Please note that the PCR Master mix
does not include ROX. The amount of ROX required is instrument dependent
and it is important to refer to the manufacturers recommendations
when deciding how much ROX to use, see Tip 8.
Step 6
Prepare reagents for
real-time PCR
Place cDNA (from Step 5), nuclease free water and PCR Master mix on
ice and thaw for 15-20 min. Protect the PCR Master mix vials from light.
Immediately before use, mix the PCR Master mix by pipetting up and
down. The rest of the reagents are mixed by vortexing and spun down.
See Tip 8
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Step 8
Real-time PCR
amplification
Perform real-time PCR amplification followed by melting curve analysis
according to Table 6.
Table 6 Real-time PCR cycle conditions
Step 9
Analyze data
Perform initial data analysis using the software supplied with the real-
time PCR instrument to obtain raw Cq values (Cp or Ct, depending on
PCR instrument). If you are using an ABI instrument, please note that
it is not recommended to use auto Ct settings. For a guide on how to
set manual baseline and threshold, refer to Tip 10, page 56 in the tips
section. Furthermore, if the data is to be analyzed using Exiqon GenEx,
the experiment must be set up as an AQ experiment, not RQ. Alternatively,
use ABI settings files available from www.exiqon.com/sds. If you are
using a Roche LC480 instrument, we recommend analysis using the
2nd derivative method.
For tips on normalization, please see Tip 11, page 58. We recommend
performing normalization and further data analysis with the Exiqon
GenEx qPCR analysis software (ww w.exiqon.com/mirna-pcr-analysis).
Please refer to our data analysis guide for recommendations.
Protocol-Hum
an,
Mouse&RatPanels
ABI instrument user?
Apply manual baseline and threshold settings! Go to www.exiqon.com/sds to download settings file
Process step Settings, LC480instrument4)
Settings, otherinstruments
Polymerase Activation /Denaturation 95C, 10 min 95C, 10 min
Amplification 45 amplificationcycles at95C, 10 s60C, 1 min,ramp-rate1.6C/s5)
Optical read
40 amplificationcycles at95C, 10 s60C, 1 min,ramp-rate1.6C/s5)
Optical read
Melting curve analysis6) Yes Yes
See Tip 11
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Protocol-Human,
Mo
use&RatPanels
2) Adjust volumes to accommodate your in-house liquid handling system and the inaccuracy these have when pipetting.3) Corresponding to 0.05ng total RNA starting material pr. PCR reaction.4) Five additional amplification cycles are required when using the LC480 instrument to allow collection of assay data
with Cp-values up to 40.5) The ramp-rate of cooling from 95C to 60C should be set to 1.6C/s. This is equivalent to 100% under standard cycling
conditions on the ABI 7500, 7900 and Viia7 instruments. If the ramp rate of cooling is too rapid, performance may be
compromised.6) Melting curve analysis of the PCR product(s) is recommended to verify specificit y and identity of the amplification
reaction. Melting curve analysis is an analysis step built into the software of instruments. Please follow the
instructions provided by the supplier. Note: The Tm of a PCR product depends on buf fer composition, salt
concentration and the PCR instrument.
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miRCURY LNA Universal RT microRNA PCR Instruction Manual
Protocol C - Focus microRNAPCR Panels
This protocol is used for conducting the first-strand cDNA synthesis and real-time PCR, using
the Cancer Focus microRNA PCR panels, 96-well plates (product numbers 203832 to 203835)
and 384-well plates (product numbers 203840 and 203841).
If working with serum plasma samples or other biofluids, please refer to the specific
miRCURY LNA Universal RT microRNA PCR Instruction Manual for biofluid samples at
www.exiqon.com/serum-plasma-pcr-manual.
Additional required materials: 96- or 384-well plate real-time PCR cycler
Thermocycler for first-strand cDNA synthesis
Micro centrifuge
Tube for mixing water and master mix (10 ml)
Sealing foils for PCR plates
Swing bucket centrifuge for 96/384-well plates
Recommended: Liquid handling robot for pipetting
Checklist: Have you considered excess volumes required for using liquid handling robotics see page 18
Did you consider how to use the RNA spike-ins please see page 15
ROX: The ExiLENT SYBR Green master mix does not include the ROX passive reference dye.
Please follow instrument manufactures recommendations
ABI instruments: The use of manual background and threshold settings is necessary for
obtaining correct PCR data. Make sure to have the optimal settings by downloading the
instrument settings file at www.exiqon.com/sds. Furthermore, if the data is to be analyzed
using GenEx, the experiment must be set up as an AQ experiment, not RQ
Have you optimized the input amount to the RT reaction in order to avoid inhibition?
Protocol-
FocusPanel
s
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Protocol-
FocusPanels
Phase I:Prepare RNA sampleSee page 52 for recommendations
Phase II: cDNA synthesisSee protocol page 38.
Phase III: real-time PCR amplificationSee protocol page 40.- Mix cDNAs with PCR Master mix- Add cDNA:PCR Master mix to
PCR plates
Phase IV: Data analysisSee data analysis guide online- Export data for further analysis- Data pre-processing, normalization
and statistical analysis
4
3
2
1
0
-1Normal Tumor Tumor
stroma
TumorTotal
miR-21let-7a
Relativeexpression
(log2)
Workflow for Focus microRNA PCR Panels (per sample)
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Protocol
The miRCURY LNA Universal RT microRNA PCR protocol is a two-part
protocol consisting of:
1. First-strand cDNA synthesis (Step 1-5)
2. Real-time PCR amplification (Step 6-10)
Important: Keep reagents and reactions on ice (or at 4C) at all times.
First strand synthesis:
Step 1
Dilute template RNA
Adjust each of the template RNA samples to a concentration of 5 ng/L
using nuclease free water.
Step 2
Prepare reagents
Gently thaw the 5x Reaction buffer and nuclease-free water, and
immediately place on ice. Mix by vortexing. Re-suspend the RNA spike-
in(s) according to the appropriate RNA Spike-in protocol (see page 15),
leave on ice for 15-20 minutes. Immediately before use, remove the
Enzyme mix from the freezer, mix by flicking the tubes and place on
ice. Spin down all reagents.
Protocol-
FocusPanel
s
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Protocol-
FocusPanels
Step 3
Combine reagents
according to Table 7
Note: remember to
calculate necessary
excess volume for
pipetting and robotic
dead volume.
When performing first-strand cDNA synthesis on multiple RNA samples,
it is recommended to prepare an RT working solution of the 5x Reaction
buffer, water, Enzyme mix and RNA spike ins (in the proportions indicated
in the first four lines of Table 7).
The following procedure is recommended:
1. Prepare the required amount of RT working solution and place it on ice.
2. Dispense RT working solution into nuclease free tubes.
3. Dispense template RNA in each tube.
Table 7 Reverse transcription reaction setup
Step 4
Mix and spin
reagents
Mix the reaction by very gentle vortexing or pipetting to ensure that all
reagents are thoroughly mixed. After mixing, spin down.
Step 5
Incubate and
heat inactivate1)
Incubate for 60 min at 42C.
Heat-inactivate the reverse transcriptase
for 5 min at 95C.
Immediately cool to 4C.
Store at 4C or freeze.
1) The protocol can be interrupted at this stage. The undiluted cDNA may be kept at -20C for up to 5 weeks (optional store
at 4C for up to 4 days). It is recommended that synthesized cDNA is stored in low-nucleic acid binding tubes or plates.
Reagent Per panel Volume (L)
5x Reaction buffer 2
Nuclease-free water 4.5
Enzyme mix 1
Synthetic RNA spike ins, optional
replace with H2O if omitted
0.5
Template total RNA (5ng/L) 2
Total volume 10
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Step 7
Combine PCR Master
mix, water and cDNA
and add to PCR plates2)
The following procedure is recommended to avoid low concentrations
of cDNA from adhering to tube surface:
1. Before removing the plate seal, briefly spin down the plate(s) in a
plate centrifuge.
2. Combine 2x PCR Master mix and water: 1000 L 2x master mix and
990 L water
(Focus panel consisting of 2x 96 assays)
3. Mix gently and spin down.
4. Add 10 L cDNA (Focus panel consisting of 2x 96 assays).
5. Add 10 L PCR Master mix: cDNA mix to each well3).
6. Seal the plate with optical sealing as recommended by the
instrument manufacturer.
The experiment can be paused at this point. Store the reactions protected
from light at 4C for up to 24 hours.
Recommendation: Include a passive reference dye in the cDNA dilution if
advised by instrument manufacturer. Please note that the PCR Master mix
does not include ROX. The amount of ROX required is instrument dependent
and it is important to refer to the manufacturers recommendations
when deciding how much ROX to use, see Tip 8.
Step 6
Prepare reagents
for real-time PCR
Place cDNA (from Step 5), nuclease free water and PCR Master mix on
ice and thaw for 15-20 min. Protect the PCR Master mix vials from light.
Immediately before use, mix the PCR Master mix by pipetting up and
down. The rest of the reagents are mixed by vortexing and spun down.
qPCR protocol:
2) Adjust volumes to accommodate your in-house liquid handling system and the losses these have when pipetting.3) Corresponding to 0.05ng total RNA starting material pr. PCR reaction.
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Step 8
Spin plate
Spin plate briefly in a plate centrifuge (1500g for 1 minute), to collect
the sample.
Step 9
Real-time PCR
amplification
Perform real-time PCR amplification followed by melting curve analysis
according to Table 8.
Table 8 Real-time PCR cycle conditions
Process step Settings, LC480instrument5)
Settings, otherinstruments4)
PolymeraseActivation/Denaturation
95C, 10 min 95C, 10 min
Amplification 45 amplificationcycles at95C, 10 s60C, 1 min,ramp-rate1.6C/s6)
Optical read
40 amplificationcycles at95C, 10 s60C, 1 min,ramp-rate1.6C/s6)
Optical read
Melting curveanalysis7)
Yes Yes
4) If using a 96-well cycler with a minimum recommended volume of 20 L (like some ABI instruments), then use 10 L
reaction volume and set the instrument settings at 20 L.5) Five additional amplification cycles is required when using the LC480 instrument to allow collection of assay data with
Cp-values up to 40.6) The ramp-rate of cooling from 95C to 60C should be set to 1.6C/s. This is equivalent to 100% under standard cycling
conditions on the ABI 7500, 7900 and Viia7 instruments. If the ramp rate of cooling is too rapid, performance may be
compromised.7) Melting curve analysis of the PCR product(s) is recommended to verify specificity and identity of the amplification reaction.
Melting curve analysis is an analysis step built into the software of instruments. Please follow the instructions provided by
the supplier. Note: The Tm of a PCR product depends on buf fer composition, salt concentration and the PCR instrument.
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Step 10
Analyze data
Perform initial data analysis using the software supplied with the real-
time PCR instrument to obtain raw Cq values (Cp or Ct, depending on
PCR instrument). If you are using an ABI instrument, please note that
it is not recommended to use auto Ct settings. Furthermore, if the data
is to be analyzed using Exiqon GenEx, the experiment must be set up as
an AQ experiment, not RQ. For a guide on how to set manual baseline
and threshold, refer to Tip 10, page 56 in the tips section. If you are
using a Roche LC480 instrument, we recommend analysis using the
2nd derivative method.
For tips on normalization, please see Tip 11, page 58. We recommend
performing normalization and further data analysis with the Exiqon
GenEx qPCR analysis software (ww w.exiqon.com/mirna-pcr-analysis).
Please refer to our data analysis guide for recommendations.
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Protocol D - Pick-&-Mix microRNAPCR Panels
This protocol is used for conducting the first-strand cDNA synthesis and real-time PCR, using
the Pick-&-Mix microRNA PCR Panel, 96-well plates (product number 203801) and 384-well
plates (product number 203802).
If working with serum plasma samples or biofluid samples, please refer to the specific
miRCURY LNA Universal RT microRNA PCR Instruction Manual for biofluid samples at
www.exiqon.com/serum-plasma-pcr-manual.
Additional required materials: 96- or 384-well plate real-time PCR cycler
Thermocycler for first-strand cDNA synthesis
Micro centrifuge
Tube for mixing water and master mix (10 ml)
Swing bucket centrifuge for 96/384-well plates.
Sealing foils for PCR plates
Recommended: Liquid handling robot for pipetting
Checklist: Have you considered excess volumes required for using liquid handling robotics
see page 18
Did you consider how to use the RNA spike-ins please see page 15
ROX: The ExiLENT SYBR Green master mix does not include the ROX passive reference dye.
Please follow instrument manufactures recommendations
ABI instruments: The use of manual background and threshold settings is necessary for
obtaining correct PCR data. Furthermore, if the data is to be analyzed using GenEx, the
experiment must be set up as an AQ experiment, not RQ. Make sure to have the optimal
settings by downloading the instrument settings file at www.exiqon.com/sds.
Have you optimized the input amount to the RT reaction in order to avoid inhibition?
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Phase I:Prepare RNA sampleSee page 52 for recommendations
Phase II: cDNA synthesisSee protocol page 46.
Phase III: real-time PCR amplificationSee protocol page 48.- Mix cDNAs with PCR Master mix- Add cDNA:PCR Master mix to primer
sets replicates (indicated by colored boxes)
Phase IV: Data analysisSee data analysis guide online- Export data for further analysis- Data pre-processing, normalization
and statistical analysis
4
3
2
1
0
-1Normal Tumor Tumor
stroma
TumorT otal
miR-21let-7a
Relativeexpression
(log2)
Workflow for Pick-&-Mix PCR plates (per sample)
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Pick-&-Mix microRNA PCR Panel layouts
Overview of the pre-defined plate layouts
available for 96-well and 384-well plates of
the Pick-&-Mix Panel. Wells in dark gray and
light greay are pre-occupied by interplate-
calibrators (UniSp3 IPC) and RNA spike-in
controls (UniSp6), respectively.
10 microRNAs x 8 samples
A
B
C
D
E
F
G
H
1 2 3 4 5 6 7 8 9 10 11 12
1 2 3 4 5 6 7 8 9 10 CP IPC
1 2 3 4 5 6 7 8 9 10 CP IPC
1 2 3 4 5 6 7 8 9 10 CP IPC
1 2 3 4 5 6 7 8 9 10 CP IPC
1 2 3 4 5 6 7 8 9 10 CP IPC
1 2 3 4 5 6 7 8 9 10 CP IPC
1 2 3 4 5 6 7 8 9 10 CP IPC
1 2 3 4 5 6 7 8 9 10 CP IPC
22 microRNAs x 4 samples
A
B
C
D
E
F
G
H
1 2 3 4 5 6 7 8 9 10 11 12
1 2 3 4 5 6 7 8 9 10 11 IPC
13 14 15 16 17 18 19 20 21 22 23
13 14 15 16 17 18 19 20 21 22 23
13 14 15 16 17 18 19 20 21 22 23
13 14 15 16 17 18 19 20 21 22 23
CP
1 2 3 4 5 6 7 8 9 10 11 IPC
CP
1 2 3 4 5 6 7 8 9 10 11 IPC
CP
1 2 3 4 5 6 7 8 9 10 11 IPC
CP
92 microRNAs x 1 sample
A
B
C
D
E
F
G
H
1 2 3 4 5 6 7 8 9 10 11 12
1 2 3 4 5 6 7 8 9 10 11 IPC
13 14 15 16 17 18 19 20 21 22 23
25 26 27 28 29 30 31 32 33 34 35
37 38 39 40 41 42 43 44 45 46 47
49 50 51 52 53 54 55 56 57 58 59
61 62 63 64 65 66 67 68 69 70 71
73 74 75 76 77 78 79 80 81 82 83
85 86 87 88 89 90 91 92 93 94 95
CP
IPC
IPC
60
72
84
96
22 microRNAs x 16 samples
A
B
C
D
E
F
G
H
I
J
K
L
M
N
O
P
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 241 2 3 4 5 6 7 8 9 10 1 1 1 2 1 3 1 4 15 1 6 1 7 1 8 19 2 0 21 2 2 C P IPC
C P I PC
C P I PC
C P I PC
C P I PC
C P I PC
C P I PC
C P I PC
C P I PC
C P I PC
C P I PC
C P I PC
C P I PC
C P I PC
C P I PC
C P I PC
1 2 3 4 5 6 7 8 9 10 1 1 1 2 1 3 1 4 1 5 1 6 1 7 1 8 1 9 2 0 21 2 2
1 2 3 4 5 6 7 8 9 10 1 1 1 2 1 3 1 4 1 5 1 6 1 7 1 8 1 9 2 0 21 2 2
1 2 3 4 5 6 7 8 9 10 1 1 1 2 1 3 1 4 1 5 1 6 1 7 1 8 1 9 2 0 21 2 2
1 2 3 4 5 6 7 8 9 10 1 1 1 2 1 3 1 4 1 5 1 6 1 7 1 8 1 9 2 0 21 2 2
1 2 3 4 5 6 7 8 9 10 1 1 1 2 1 3 1 4 1 5 1 6 1 7 1 8 1 9 2 0 21 2 2
1 2 3 4 5 6 7 8 9 10 1 1 1 2 1 3 1 4 1 5 1 6 1 7 1 8 1 9 2 0 2 1 2 2
1 2 3 4 5 6 7 8 9 10 1 1 1 2 1 3 1 4 1 5 1 6 1 7 1 8 1 9 2 0 2 1 2 2
1 2 3 4 5 6 7 8 9 10 1 1 1 2 1 3 1 4 1 5 1 6 1 7 1 8 1 9 2 0 2 1 2 2
1 2 3 4 5 6 7 8 9 10 1 1 1 2 1 3 1 4 1 5 1 6 1 7 1 8 1 9 2 0 2 1 2 2
1 2 3 4 5 6 7 8 9 10 1 1 1 2 1 3 1 4 1 5 1 6 1 7 1 8 1 9 2 0 2 1 2 2
1 2 3 4 5 6 7 8 9 10 1 1 1 2 1 3 1 4 1 5 1 6 1 7 1 8 1 9 2 0 2 1 2 2
1 2 3 4 5 6 7 8 9 10 1 1 1 2 1 3 1 4 1 5 1 6 1 7 1 8 1 9 2 0 2 1 2 2
1 2 3 4 5 6 7 8 9 10 1 1 1 2 1 3 1 4 1 5 1 6 1 7 1 8 1 9 2 0 2 1 2 2
1 2 3 4 5 6 7 8 9 10 1 1 1 2 1 3 1 4 1 5 1 6 1 7 1 8 1 9 2 0 2 1 2 2
1 2 3 4 5 6 7 8 9 10 1 1 1 2 1 3 1 4 1 5 1 6 1 7 1 8 1 9 2 0 2 1 2 2
46 microRNAs x 8 samples
A
B
C
D
E
F
G
H
I
J
K
L
M
N
O
P
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24
1 2 3 4 5 6 7 8 9 10 11 1 2 13 14 1 5 16 1 7 18 19 20 21 22 23 CP
25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47
1 2 3 4 5 6 7 8 9 10 11 1 2 13 14 1 5 16 1 7 18 19 2 0 21 2 2 2 3
25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47
1 2 3 4 5 6 7 8 9 10 11 1 2 13 14 1 5 16 1 7 18 19 2 0 21 2 2 2 3
25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47
1 2 3 4 5 6 7 8 9 10 11 1 2 13 14 1 5 16 1 7 18 19 2 0 21 2 2 23
25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47
1 2 3 4 5 6 7 8 9 10 11 1 2 13 14 1 5 16 1 7 18 19 2 0 21 2 2 23
25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47
1 2 3 4 5 6 7 8 9 10 11 1 2 13 14 1 5 16 1 7 18 19 2 0 21 2 2 23
25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47
1 2 3 4 5 6 7 8 9 10 11 1 2 13 14 1 5 16 1 7 18 1 9 2 0 21 2 2 23
25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47
1 2 3 4 5 6 7 8 9 10 11 1 2 13 14 1 5 16 1 7 18 1 9 2 0 21 2 2 23
25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47
IPC
CP
IPC
CP
IPC
CP
IPC
CP
IPC
CP
IPC
CP
IPC
CP
IPC
94 microRNAs x 4 samples
A
B
C
D
E
F
G
H
I
J
K
L
M
N
O
P
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 241 1 22 33 44 55 66 77 88 99 1010 1111 1212
1 1 22 33 44 55 66 77 88 99 1010 1111 1212
13 13 1414 1515 1616 1717 1818 1919 2020 2121 2222 2323 2424
13 13 1414 1515 1616 1717 1818 1919 2020 2121 2222 2323 2424
25 25 2626 2727 2828 2929 3030 3131 3232 3333 3434 CPCP 3636
25 25 2626 2727 2828 2929 3030 3131 3232 3333 3434 CPCP 3636
37 37 3838 3939 4040 4141 4242 4343 4444 4545 4646 4747 4848
37 37 3838 3939 4040 4141 4242 4343 4444 4545 4646 4747 4848
49 49 5050 5151 5252 5353 5454 5555 5656 5757 5858 5959 6060
49 49 5050 5151 5252 5353 5454 5555 5656 5757 5858 5959 6060
61 61 6262 6363 6464 6565 6666 6767 6868 6969 7070 IPCIPC 7272
61 61 6262 6363 6464 6565 6766 6767 6868 6969 7070 IPCIPC 7272
73 73 7474 7575 7676 7777 7878 7979 8080 8181 8282 8383 8484
73 73 7474 7575 7676 7777 7878 7979 8080 8181 8282 8383 8484
85 85 8686 8787 8888 8989 9090 9191 9292 9393 9494 9595 9696
85 85 8686 8787 8888 8989 9090 9191 9292 9393 9494 9595 9696
380 microRNAs x 1 sample
A
B
C
D
E
F
G
H
I
J
K
L
M
N
O
P
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 241 2 43 65 87 109 1211 1413 1615 1817 2019 2221 2423
25 26 2827 3029 3231 3433 3635 3837 4039 4241 4443 4645 4847
5049 51 5352 5554 5756 5958 6160 6362 6564 6766 6968 7170 72
73 74 7675 7877 8079 8281 8483 8685 8887 9089 9291 9493 IPC95
97 98 10099 102101 104103 106105 108107 110109 112111 114113 116115 118117 120119
121 122 124123 126125 128127 130129 132131 134133 136135 138137 140139 142141 144143
145 146 148147 150149 152151 154153 156155 158157 160159 162161 164163 166165 168167
169 170 172171 174173 176175 178177 180179 182181 184183 186185 188187 190189 IPC191
193 194 196195 198197 200199 202201 204203 206205 208207 210209 212211 214213 216215
217 218 220219 222221 224223 226225 228227 230229 232231 234233 236235 238237 240239
241 242 244243 246245 248247 250249 252251 254253 256255 258257 260259 262261 264263
265 266 268267 270269 272271 274273 276275 278277 280279 282281 284283 286285 CP287
289 290 292291 294293 296295 298297 300299 302301 304303 306305 308307 310309 312311
313 314 316315 318317 320319 322321 324323 326325 328327 330329 332331 334333 336335
337 338 340339 342341 344343 346345 348347 350349 352351 354353 356355 358357 360359
361 362 364363 366365 368367 370369 372371 374373 376375 378377 380379 382381 IPC383
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Protocol
The miRCURY LNA Universal RT microRNA PCR protocol is a two-part
protocol consisting of:
1. First-strand cDNA synthesis (Step 1-5)
2. Real-time PCR amplification (Step 6-11)
Important: Keep reagents and reactions on ice (or at 4C) at all times.
First strand synthesis:
Step 1
Dilute template RNA
Adjust each of the template RNA samples to a concentration of 5 ng/L
using nuclease free water.
Step 2
Prepare reagents
Gently thaw the 5x Reaction buffer and nuclease-free water, and
immediately place on ice. Mix by vortexing. Re-suspend the RNA spike-
in(s) according to the appropriate RNA Spike-in protocol (see page 15),
leave on ice for 15-20 minutes. Immediately before use, remove the
Enzyme mix from the freezer, mix by flicking the tubes and place on
ice. Spin down all reagents.
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Step 3
Combine reagents
according to Table 9
When performing first-strand cDNA synthesis on multiple RNA samples,
it is recommended to prepare an RT working solution of the 5x Reaction
buffer, water, Enzyme mix and RNA spike ins (in the proportions indicated
in the first four lines of Table 9).
The following procedure is recommended:
1. Prepare the required amount of RT working solution and place it on ice.
2. Dispense RT working solution into nuclease free tubes.
3. Dispense template RNA in each tube.
Table 9 Reverse transcription reaction setup per sample1)
The amount of 100x fold diluted cDNA needed for the different
Pick-&-Mix layouts can be seen in Table 10 Step 7.
Reagent Volume (L)< 100 miRNAanalyzed persample
Volume (L)> 100 miRNAanalyzed persample
5x Reaction buffer 2 4
Nuclease-free water 4.5 9
Enzyme mix 1 2
Synthetic RNA spike ins, optionalreplace with H
2O if omitted
0.5 1
Template total RNA(5ng/L)
2 4
Total volume 10 20
Step 4
Mix and spin reagents
Mix the reaction by very gentle vortexing or pipetting to ensure that all
reagents are thoroughly mixed. After mixing, spin down.
1) The amount of cDNA required per sample depends on the number of microRNAs analyzed per sample. The volumes
suggested here provides excess amount of cDNA, which ensures the highest possible reproducibility because these
volumes can be pipetted with great accuracy.
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2) Although not recommended, the protocol can be interrupted at this stage. The undiluted cDNA may be kept at -20C for
up to 5 weeks (optional store at 4C for up to 4 days). It is recommended that synthesized cDNA be stored in low-nucleic
acid binding tubes or plates.
Step 5
Incubate and
heat inactivate2)
Incubate for 60 min at 42C.
Heat-inactivate the reverse transcriptase
for 5 min at 95C.
Immediately cool to 4C.
Store at 4C or freeze.
qPCR protocol:
Step 6
Prepare reagents
for real-time PCR
Place cDNA (from Step 5), nuclease free water and PCR Master
mix on ice and thaw for 15-20 min. Protect the PCR Master mix
vials from light. Immediately before use, mix the PCR Master mix
by pipetting up and down. The rest of the reagents are mixed by vortexing
and spun down.
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Step 7
Dilute cDNA template
100x in nuclease
free water3)
Dilute the cDNA from the RT reactions to give a final 100x dilution.
Suggested cDNA dilution procedures is shown in Table 10 along with
required volumes of diluted cDNA for the different Pick-&-Mix pre-
defined layouts. It is receommeded that low-nucleic acid binding.
For fully customized layouts dilutions may be adjusted to the specific
replicate scheme. tubes or plates are used. It is not recommended to
store the 1:100 dilution of cDNA.
Table 10 Amount of 100x diluted cDNA needed in Pick-&-Mix plates
Numbers in parenthesis designate total number of assays/sample,
including controls and inter-plate calibrators. Loss from pipetting is not
included in the volumes. listed in the right column (diluted cDNA needed).
Recommendation: Include a passive reference dye in the cDNA dilution if
advised by instrument manufacturer. Please note that the PCR Master mix
does not include ROX. The amount of ROX required is instrument dependent
and it is important to refer to the manufacturers recommendations
when deciding how much ROX to use, see Tip 8.
Pick-&-Mix plateconfiguration
Suggested cDNAdilution procedurecDNA + nucleasefree water (L)
Volume (L) of dilutedcDNA needed for eachPick-&-Mix plate
8x10 (12) 2 + 198 60
4x22 (24) 2 + 198 120
1x92 (96) 5 + 495 480
16x22 (24) 2 + 198 120
8x46 (48) 3 + 297 240
4x94 (96) 5 + 495 4801x380 (384) 20 + 1980 1920
3) Adjust volumes to accommodate your in-house liquid handling system and the losses these have when pipetting.
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Step 8
Combine cDNA and
PCR Master mix 1:1
and add to PCR plates
The following procedure is recommended:
1. Before removing the plate seal, briefly spin down the plate(s) in a
plate centrifuge.
2. Combine 2x PCR Master mix and 100x diluted cDNA 1:1 (e.g. 500 L
2x PCR master mix and 500 L diluted cDNA).
3. Mix gently by inverting the tube, spin down.
4. Add 10 L PCR Master mix:
cDNA mix to each well.4)
5. Seal the plate with optical sealing as recommended by the
instrument manufacturer.
The experiment can be paused at this point. Store the reactions protected
from light at 4C for up to 24 hours.
Step 9Spin plate
Spin plate briefly in a plate centrifuge (1500g for 1 minute), to removeair bubbles.
Step 10
Real-time
PCR amplification
Perform real-time PCR amplification followed by melting curve analysis
according to Table 11.
Table 11 Real-time PCR cycle conditions
Process step Settings, LC480
instrument
5)
Settings, other
instruments
6)
Polymerase Acti vation/Denaturation 95C, 10 min 95C, 10 min
Amplification 45 amplificationcycles at95C, 10 s60C, 1 min,ramp-rate1.6C/s7)
Optical read
40 amplificationcycles at95C, 10 s60C, 1 min,ramp-rate1.6C/s7)
Optical read
Melting curveanalysis8)
Yes Yes
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Step 11
Analyze data
Perform initial data analysis using the software supplied with the real-
time PCR instrument to obtain raw Cq values (Cp or Ct, depending on
PCR instrument). If you are using an ABI instrument, please note that
it is not recommended to use auto Ct settings. For a guide on how to
set manual baseline and threshold, refer to Tip 10, page 56 in the tips
section. Furthermore, if the data is to be analyzed using Exiqon GenEx,
the experiment must be set up as an AQ experiment, not RQ.
For tips on normalization, please see Tip 11, page 58. If you are using
a Roche LC480 instrument, we recommend analysis using the 2nd
derivative method. We recommend performing normalization and
further data analysis with the Exiqon GenEx qPCR analysis software
(ww w.exiqon.com/mirna-pcr-analysis). Please refer to our data analysis
guide for recommendations.
4) Corresponding to 0.05ng total RNA starting material pr. PCR reaction.5)Five additional amplification cycles are required when using the LC480 instrument to allow collection of assay data with
Cp-values up to 40.6)If using a 96-well cycler with a minimum recommended volume of 20 L (like some ABI instruments), then use 10