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Effect of RNA integrity on real-time PCR results Tips to achieve a true RNA profiling suitable for real-time PCR studies

Dr. Ina Scheuerpflug, Ph.D.

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Practical Hints for RT-PCR 2

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Critical steps for real-time PCR analysis

RNA critical steps

• Principles of RNA stabilization

• Critical factors for RNA purification

• Choosing the right RNA Purification method

• microRNAs - micromanagers of gene expression

Improved methods for cDNA synthesis

Practical Hints for Real-Time PCR

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DNA

DNA

mRNA

TranscriptionNucleus

Cytoplasm

tRNAs

Attachedamino acid

Growingpeptide

mRNA

Function and abundance of RNA

RNA species Relative abundance

rRNA 80-85%

tRNAs, snRNAs (<120 nt)

15-20%

mRNA 1-5%

Practical Hints for Real-Time PCR 4

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8Critical steps:

• Stabilization of RNA patterns and RNA quality

• RT enzyme efficiency

• Priming strategy

• Genomic DNA contaminations

Critical steps for real-time PCR analysis

5Practical Hints for Real-Time PCR

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What happens to RNA ex vivo?

mR

NA

copy

num

ber

Blood collectionTime

Induction

In vivo transcript level at to

Cell Death/Enzymatic Degradation

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Principles of RNA stabilization

Requirements:• Stop induction of new RNA transcription (e.g., stress response)

• Prevent regulated turnover of mRNA

• Prevent unspecific degradation of RNA, either enzymatically (due to release of nucleases from specific

cell types or compartments), or chemically (pH, temperature, etc. dependent)

Approaches:• Agents that bind directly to nucleic acids

• Agents that lyse cells

• Agents that inhibit, denature and/or precipitate nucleases (proteins in general)

Fixation:• Preserve morphology (usually with no consideration of effects on RNA)

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Requirements for stabilization of blood and tissue samples

Due to the differences of the sample materials, blood and tissue samples have different requirements concerning the appropriate stabilization technology.

Blood contains only solitary cells but has a high protein content:• Diffusion rates of stabilizing agent no problem• Agents that precipitate or cross-link proteins inappropriate• Blood volume is not limited in principle (container is limited)• One-step stabilization technology required

Tissue contains connected cells:• Diffusion rates of the stabilizing agent are critical• Thickness of the tissue sample is limited • Multi-step technology applicable as tissue piece transferrable• Protein precipitation or cross-link is not a problem in principle

8Practical Hints for Real-Time PCR

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GITC temporarily denatures

Native RNase

BME permanently denatures

• Chaotropic salt

• Reducing agents

• Inhibitors

• Extreme cold

• Stabilizers

Degradation of RNA by RNases - RNAse denaturing agents

9Practical Hints for Real-Time PCR

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Different collection and stabilization solutions for different sample types

10Practical Hints for Real-Time PCR

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Critical steps for real-time PCR analysis

RNA critical steps

• Principles of RNA stabilization

• Critical factors for RNA purification

• Choosing the right RNA Purification method

• microRNAs - micromanagers of gene expression

Improved methods for cDNA synthesis

Practical Hints for Real-Time PCR

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Critical factors for RNA purification

Quality of the purified RNA: integrity and purity• Quality of starting sample material• Inactivation of RNases• Co-purification of potential inhibitors e.g., for the RT-step• Protein (nuclease) contamination• gDNA contamination

RNA yield:• Efficient cell/tissue lysis• mRNA content of total RNA is only 1-5%• Purification of total RNA and small RNA (e.g., miRNA)

Practical Hints for Real-Time PCR 12

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Silica-Technology: Components 1 - Lysis

Chaotropic Reagents:

• Guanidine hydrochloride (GuHCI)

• Guanidine isothiocyanate (GITC)

• Urea

• Sodium perchlorate (NaClO4)

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Silica-Technology: Components 2

Silica:

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Silica Technology: 1. Nucleic Acid in Solution

DNA

Water Molecules

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Silica Technology: 2. Effect of Chaotropic Salts

Chaotropic Salt

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Silica Technology: 3. Binding of Nucleic Acid

Silica

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Silica Adsorption

Nucleic acid (aqueous)

Nucleic acid (bound to silica)

Adsorption

Chaotropic saltAlcoholLow pH

Elution

Low saltHigh pH

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Lysis

Sample

Solution: Different lysis steps for different material

Lysis: Protocol dependent on sample type

• Mechanical disruption of cells/tissue (e.g., TissueLyser)

• Lysis of cells and nuclei by chaotropic salts

• Inactivation of RNases by chaotropic salts in lysis buffer

• Proteinase for lysis of tough samples (e.g., muscle)requires conditions where proteinase is active but RNases are not

• QIAzol (phenol-guanidine based):lysis of tough samples, and removal of contaminants by organic extractionvery versatile, but more work, hazardous reagent

Practical Hints for Real-Time PCR 19

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Bind Wash EluteLysis

Sample

Silica Adsorption Protocol

Lysis: • Protocol dependent on the sample type

Bind nucleic acids to silica: • High concentrations of chaotropic salts, low pH, + alcohol

Wash the silica membrane: • Removal of proteins, inhibitors and chaotropic salts

Elute nucleic acids from silica membrane: • Water or low-salt buffer pH 7–8

Ready-to-usepure NA

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Effect of phenol on UV absorbance

Wavelength [nm] Wavelength [nm]

[Abs]

0.4

0.0 0.0

0.3

220 220320 320

Phenol in Water

[Abs]

260 280 260 280

RNA contaminatedwith phenol (1/100,000)

Pure RNA

Phenol (1/5000)

Phenol contamination imitates higher RNA content of the sample

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RNeasy

mRNA enrichment

Selective exclusion of RNA < 200 nt

mRNA

rRNA > 200 nt

tRNA, snRNA, miRNA < 200 nt

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Choosing the right RNA Purification product / protocol

Is the RNA purification procedure appropriate for the amount of starting material?• Overloading usually results in lower purity, lower yields• With small samples it is important to use an efficient RNA purification method

RNA purification from large samples: • Provides a pool of RNA that can be used for multiple experiments• May be required to get sufficient amounts of RNA from samples that have low

RNA content• High binding capacity – usually also implies higher dead volume

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RNeasy plus concept

Upgraded RNeasy Kit• Additional gDNA Eliminator columns• New RLT Plus Lysis Buffer

For purification of total RNA from animal cells and easy-to-lyse tissues, with gDNA Eliminator columns included

Alternative to on-column DNAse digest – similar efficiency, but faster, more convenient, and cheaper

NOT an alternative for difficult samples were on-column DNase digest fails

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RNeasy plus – Linearity and Purity

• DNA removal efficiency optimal for ≤20 µg gDNA (binding capacity >100 µg)

• High RNA yield - excellent linearity down to 100 cells

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gDNA contamination:

RNA conc.:

~150 ng /µl – lung

~500 ng/µl - kidney

~900 ng/µl - liver

RNeasy plus – Tissue

• Example: 10 mg rat tissue per prep• Up to 1000x less gDNA, dep. on tissue

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New RNeasy development: RNeasy Plus Universal

• For purification of total RNA from any type of tissue using gDNA Eliminator solution

• One-for-all tissue solution• Time-saving integration of RNeasy and QIAzol technologies• Fast non-enzymatic removal of genomic DNA• Pure RNA for use in all downstream applications

• Optimized protocols enable purification of high-quality RNA from any type of tissue, even difficult-to-lyse tissues.

• QIAzol Lysis Reagent included for lysing fatty tissue and other types of tissue, and RNeasy spin columns for purifying high-quality RNA.

• Kits are available in two formats: the RNeasy Plus Universal Mini Kit enables purification of RNA from up to 50 mg tissue and the RNeasy Plus Universal Midi Kit enables purification of RNA from up to 250 mg tissue.

• The RNeasy Plus Universal Mini Kit can be automated on the QIAcube.

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Upgrade your expectations! RNeasy Plus Universal

QIAzol + gDNA Eliminator Solution + RNeasy Mini or Midi

Add gDNA Eliminator

Solution and chloroform and

shake

One-for-all tissue kit with efficient gDNA Eliminator solution

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Formaldehyde-fixed paraffin-embedded samples - Challenges

Formaldehyde fixation results in:

• Protein-protein cross-linking• Protein-nucleic acid cross-linking• Nucleic acid-nucleic acid cross-linking

Paraffin embedding means:

Extended incubation at >60°C results in nucleic acid fragmentation

Deparaffination is required prior to nucleic acid preparation

Harsh lysis conditions are required to break up tissue

Fragmentation of nucleic acids

Remaining formaldehyde modifications on nucleic acids interfere with enzymatic assays (reverse transcription, PCR)

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RNeasy FFPE

Optimized deparaffination and lysis• Enabling efficient RT-PCR by breaking formaldehyde crosslinks

• RNA preservation

gDNA eliminator spin column• Easy and efficient gDNA removal without compromising RNA

yield

• Faster and more economical than DNase digestion

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Heat incubation

Heat incubation in Prot.K digestion buffer helps to break formaldehyde crosslinks

• shorter PK digest possible (15 min)

• better yield

• better substrate for PCR

Limited by RNA stability:

longer incubation / higher temp. will remove more of the formaldehyde modifications, but also results in more RNA fragmentation

(1 section, 10 µm per prep; brain samples are from a different experiment)

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Recovery of All Usable RNA

Small RNA fragments efficiently recovered with RNeasy FFPE

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RNA purified from 6-month-old FFPE rat liver using the RNeasy FFPE Kit or a kit from Supplier R was analyzed on the Agilent 2100® bioanalyzer

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RNA from FFPE sections: quality / integrity

Quality of RNA from FFPE samples is compromised due to:• Fragmentation (heat + buffer conditions)

Formaldehyde crosslink / modification of RNA

For cDNA synthesis and PCR:• avoid oligo-dT priming (better: random or gene-specific priming)• choose short amplicons (<500 nt if possible)

18

S

28

S

Flu

or

es

ce

nc

e

Time (seconds)

0

10

20

30

40

50

60

19 24 29 34 39 44 49 54 59 64 69

Flu

or

es

ce

nc

e

Time (seconds)

0

10

20

30

40

50

60

70

19 24 29 34 39 44 49 54 59 64 69

Fresh FFPE sample ~6 mo. old FFPE samplePCRcontrol

128 208404

668

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Fast RNeasy FFPE Procedure

No need for extra DNAse digest

Lysis with Prot. K digestion in only 15 min.

In only 70 min all usable RNA.

80°C, 15 min. to reverse formalin crosslinking

14-30 μl elution vol.

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AllPrep solution: One Sample – Two Analytes

15-30 kb DNA

Total RNA Genomic DNA

RIN 10 RNA

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Total RNA and genomic DNA were purified from 10 mg samples of rat liver using the AllPrep DNA?RNA Mini Kit. A. Purified RNA samples were analyzed on a 1.2% formaldehyde agarose gel. B. Purified DNA samples were analyzed on a 0.8% agarose gel. M: markers

Total RNA was purified from 1 x 106 Jurkat cells using the AllPrep DNA/RNA Mini Kit and analyzed on the Agilent 2100 bioanalyzer. The RIN value was a maximum of 10, indicating intact RNA. M: markers; 18S: 18S rRNA; 28S: 28S rRNA

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Short Streamlined Procedure for DNA/RNA Purification

Novel AllPrep DNA column

Bind – Wash - Elute

DNA and RNA in 35 minutes

RNeasy column

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Practical Hints for Real-Time PCR 36

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Critical steps for real-time PCR analysis

RNA critical steps

• Principles of RNA stabilization

• Critical factors for RNA purification

• Choosing the right RNA Purification method

• microRNAs - micromanagers of gene expression

Improved methods for cDNA synthesis

Practical Hints for Real-Time PCR

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microRNAs - micromanagers of gene expression

Characteristic of microRNAs• Naturally occurring, endogenous small RNA

• A mature miRNA is approximately 22 nt long

• Regulate at least 1/3 of the protein encoding genes

• > 500 microRNAs in human

• Mediate Post Transcriptional Gene Silencing either by translational repression or target mRNA degradation

• A typical human cell harbors 1.000 to 200.000 miRNAs in patterns unique to particular cell types

• One miRNA might bind 100 or more target mRNAs

• A single mRNA might have target sites for several different miRNAs

• http://microrna.sanger.ac.uk

Fine-tuning of gene expression • Cell fate

• Differentiation

• Morphogenesis

• Development

• Many aspects of physiology

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Biogenesis of microRNA

• Transcribed by RNA Polymerase II as - pri-miRNAs

• In the nucleus, Pri-miRNAs are processed to ~ 70 nt hairpin-like pre-miRNAs by Drosha

• Pre-miRNAs are then exported by Exportin 5

• In the cytosol pre-miRNAs are processed to mature miRNAs by Dicer

• These miRNAs are incorporated into the RISC (RNA-Induced Silencing Complex)

• miRNAs with imperfect base pairing to the target mRNA• lead to translational repression and/or mRNA

degradation

Cytosol

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RIBOSOME

miRNA - How It Works

3`5` AAAAAmRNA

RISC like complex 5`P3`OH

5`P3`OH

3`UTR3`UTR Seed Region match

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• miRNA is incorporated in RISC and binds to a 7-9 nucleotide region in the 3´untranslated region of the mRNA (= 3´UTR seed region)

• This binding prevents the Ribosome from translation of the GOI.→ The protein is down regulated

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miRNAs will likely enter the clinic as biomarkers & diagnostics

• Most are expected to be in oncology because many miRNAs affect the cell cycle

• Half of all human miRNAs discovered so far are expressed abnormally in at least 1 cancer

• Some miRNA profiles are specific to one cancer, others are common to many cancers

Regardless of whether the change in expression is ‘cause’ or ‘downstream effect’, miRNA profiling is helping discover new markers

for human disease classification.

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miRNeasy - copurification or enrichment of miRNA

Starting samples:• All types of tissues and cells• White blood cells• FFPE with miRNeasy FFPE Kit

Flexible protocols:• Copurification of miRNA & total RNA• miRNA enriched fraction and total RNA

> 200nt

Formats:• Single spin• 96-well plate• Manual or automated on QIAcube

Principle:• Proven silica membrane technology

Products:• miRNeasy Mini Kit• miRNeasy 96 Kit• miRNeasy FFPE Kit

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Efficient copurification from wide array of tissues

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RNA isolation kit selector wheel

wheel.exe

• If you would like to order the wheel for your lab just send us an email• Download the selection wheel at: www.QIAGEN.com

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RNA resource center

https://www.QIAGEN.com/qdm/rna/resources/

Practical Hints for Real-Time PCR 45

Sample to Insight

Critical steps for real-time PCR analysis

RNA critical steps

• Principles of RNA stabilization

• Critical factors for RNA purification

• Choosing the right RNA Purification method

• microRNAs - micromanagers of gene expression

Improved methods for cDNA synthesis

Practical Hints for Real-Time PCR

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Sample to Insight

Critical factors for an efficient RT step

RT enzyme efficiency is influenced by:

• Quality of the RNA starting material• Phenol contamination• Alcohol contamination• Salt contamination• Protein inhibitors

• Affinity to the RNA to avoid secondary structure effects• Choice of priming method

• Random priming vs. Oligo dT priming• Genomic DNA contamination• Sequences near the 5‘end affect the cDNA yield• Use of internal controls is critical

Practical Hints for Real-Time PCR 47

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High efficiency and sensitivity – Why?

• QuantiNova Reverse Transcriptase

• Highly efficient transcription for sensitive detection even of low abundance targets

• Novel Reverse Transciptase Use of a wide range of RNA amounts (10 pg – 5 μg)

• High affinity for RNA – leads to high sensitivity

• Up-scaling option for larger input volumes (up to double volume)

• Successful use even for difficult templates

• Plus RNase inhibitor

• gDNA removal buffer

• Efficient removal of gDNA (>1000 fold reduction) is integrated in the protocol

• RT-Primer Mix

• Optimized mix of oligo-dT and random primers

• Internal control

• Optional use of internal control included to monitor cDNA synthesis efficiency

Practical Hints for Real-Time PCR 48

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QuantiNova Reverse Transcription Kit - Protocol

• With unique Internal Control RNA• With gDNA removal

Fast and convenient protocol 20 min:

1. RNA (potentially contaminated with gDNA)Add gDNA Removal Buffer (incl. RNase Inhibitor)Optionally spike in Internal Control RNA2 min 45°C

2. Synthesize cDNAAdd 5 µl RT-Master Mix 3 min 25°C, 10 min 45°C, 5 min 85°CcDNA (incl. IC cDNA, if applicable) Stable storable, colorable with QN Yellow Template Dye

3. Stop reaction (95°C, 3 min) get cDNA without gDNA contamination!!!

cDNA synthesis procedure in only 20 min

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QuantiNova Reverse Transcription Kit – Internal Control

QuantiNova IC reliably indicates inhibition or failure of RT or qPCR reaction.

Is a synthetic RNA that can be optionally used to monitor succesful RT.

Different amounts of SDS spiked into the PCR reaction • The IC indicated inhibition by delayed CT valuesReliable in-process monitoring of RT and qPCR performance

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QuantiNova Reverse Transcription Kit – gDNA Removal

Efficient, optional gDNA removal prevents CT shifts caused by DNA contamination

Precise mRNA quantification even if exon spanning primers cannot be used

10 ng gDNA in PCR reaction as positive control

100 & 10 ng gDNA spiked in without gDNA removal in RT-rxn

NTC

100 & 10 ng gDNA spiked in with gDNA removal

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Effect of primer choice

Amplicon – 3‘-end: 2 kb(Mix or Oligo-dT)

(N)x Oligo-dT+(N)x

Oligo-dT

Amplicon – 3‘-end: 6 kb

(Mix or Oligo-dT)

Oligo-dT

Oligo-dT+(N)x

(N)x

810 kb transcript (amplicon 2kb and 6kb away from 3´end)

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• High sensitivity due to novel enzyme

• Synthesize cDNA and remove genomic DNA in 20 min

• Integrated genomic DNA removal step

• Reverse transcription from all regions due to primer Mix (5’region)

• Internal control

Summary: QuantiNova Reverse Transcription Kit

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You can find all product information at:

https://www.qiagen.com/de/products/life-science-research/cancer-research/gene-expression-analysis

/

For additional information

Practical Hints for Real-Time PCR 54

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Thank you for attending today’s webinar!

Contact QIAGEN Technical ServiceCall: 1-800-426-8157 for US

Call: +49 2103-29-12400 for EU

Email: techservice-na@qiagen.com

techservice-eu@qiagen.com

Ina Scheuerpflug, Ph.D.Ina.Scheuerpflug@qiagen.com

Questions?

Thank you for attending