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Gene expression detection methods RNA
Dr. Ingrid Müller - AG von Laer
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TRANSCRIPTIONAL CONTROL REGULATES DIFFERENTIATION
Four different human cells - same genes, different structures and functions due todifferential gene expression
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THE CENTRAL DOGMA
• Flow of Information: DNA Replication
Transcription Translation
A B
Cells in all living organisms are continually activating or deactivatinggenes through gene expression, which contain the information requiredfor producing proteins through proteins synthesis. When a particularprotein is required by the cell, the gene coding for that protein isactivated. The first stage in producing a protein involves the productionof an RNA copy of the gene's DNA sequence. This RNA copy is themessenger RNA. The amount of mRNA produced correlates with theamount of protein eventually synthesised and measuring the amount ofa particular mRNA produced by a given cell or tissue is often easierthan measuring the amount of the final protein. Levels in geneactivation may vary between cancerogenic and healthy cells.
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MOLECULAR METHODS TOOL BOX
•Knock-out•Integrational mutagenesis•Classic genetics
•(microinjection)•Electroporation,•lipofection,•transgenics
•Reporter genes
DNA(gene)
•RNAi &•siRNA•Morpholinos
•microinjection•Electrophoresis•Northern Blot•RNAse protectionassays•Microarrays•RT-PCR•RNA in-situ
RNA
•pharmacological inhibitors,•dominant-negativeproteins,• protein depletion usingantibodies
•(adding proteins to invitro reactions)
•Western Blot•Proteomics•immuno-histochemisty•protein chimera
Protein
III. InhibitionII. OverexpressionI. Analysis
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RNA METHODS: ELECTROPHORESIS
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PURIFICATION OF MESSENGER RNA USING OLIGO DTCOLUMNS
Oligo(dT) attached to cellulose
Total cellular RNA;apply at room temperature toanneal polyA tail to oligo(dT)
} non-polyARNA flowsthrough
AAAA..TTTT
polyA binds to oligo(dT) oncolumn
65oC
TTTT
AAAAA..
polyA mRNA elutes at high temperature
Isolate RNA
Break opencells in thepresence of
RNAseinhibitors
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RNA FRAGMENTS SEPARATED BY GEL ELECTROPHORESIS
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RNA METHODS: ELECTROPHORESIS
Detection using SYBR Green II:staining of ss nucleic fragments
• Denaturing of RNA to break upsecondary structures
• TAE / TBE running buffer
• Agarose / Polyacrylamide gels
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• Isolated mRNA separated on gel according to size• mRNA transferred to a membrane and hybridized with smallnumber (1-5) of radioactively labeled DNA/RNA probes (35S or 32P)• Probe corresponds to gene of interest• Target RNA is spatially fixed and the labeled probe is in solution
• Reverse northern blot:Probe: isolated mRNASubstrate (fixed to membrane): DNA/RNA fragments
NORTHERN BLOTS
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NORTHERN BLOT
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NORTHERN BLOT
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NORTHERN BLOT
target gene 10x
internal control geneactin, GAPDH, RPLP0 etc. 2x
Ratio target gene in experimental/control = fold change in target gene fold change in reference gene
control sample
Corrected fold increase = 10/2 = 5
How can amounts of RNA be quantified? This slide shows a virtual Northernwith two lanes, one with RNA from control cells, the other with RNA from theexperimental sample (eg drug treated cells). Let’s say that there is 10x theamount of signal in the experimental sample compared to the control samplefor the target gene. This could mean expression of the gene has increased 10-fold, or it could mean that there is 10x as much RNA in the expt lane. Tocheck for this one usually does a so-called ‘loading control’ in which the blotis probed for expression of a gene which does not change (e.g. actin, GAPDH,cyclophilin, RPLP0 mRNAs; ribosomaL RNA). In this case, let’s say that theloading control shows that there is twice as much RNA in the expt lane. Thusthe real change in the target gene is 10/2 =5 fold. We can express this in amore general fashion:rat io targ et g ene ( experim ental/contro l) = fo ld chang e in targ et g ene ( expt/contro l)
fo ld chang e inreference g ene ( expt/contro l)
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NORTHERN BLOT
Measure relative expression levels of mRNA
1. mRNA isolation and purification2. electrophorese on a gel3. The gel is probed by hybridizing with a labeled clone for the geneunder study.
northern blotting of human tissues
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Pros– Established and widely accepted method for single mRNA
species detection– Newer techniques can be done without radiation
(fluorescence)
Cons– Same semi-quantitative limitations as seen in SDS-
PAGE/Western Blots for protein– Time consuming– Low throughput
RNA METHODS: NORTHERN PRO’S AND CON’S
Perform 35,000 Northerns to monitorexpression of all genes!!!
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MICROARRAYS
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FINGERPRINTS OF GENE EXPRESSION ⇒ MICROARRAYS
Normal Cell
Cancer Cell
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MICROARRAY
Collection of microscopic DNA spots attached to a solidsurface (glass, plastic, silicon chip) forming an array forthe purpose of expression profiling, monitoring expressionlevels for thousands of genes simultaneously
Applications:Analysis of expression patterns in:
– Tissues– Disease states
Sub-typing complex genetic diseasee.g. cancer
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DNA ARRAY TECHNOLOGY
Array TypeSpot Density
(per cm2)
Probe Target Labeling
Nylon Macroarrays < 100 cDNA RNA Radioactive
Nylon Microarrays < 5000 cDNA mRNA Radioactive/Flourescent
Glass Microarrays < 10,000 cDNA mRNA Flourescent
Oligonucleotide Chips <250,000 oligo's mRNA Flourescent
Fabrication
Printing using fine-pointed pins on glass slide
Photolithography
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PHYSICAL SPOTTING
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PHOTOLITHOGRAPHIC SPOTTING
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TWO POPULAR MICROARRAYING PLATFORMS
Spotted microarrays
Probes:Synthesized oligos (70 mer)cDNASmall PCR products (500-1,000bp)Corresponding to mRNAs>10,000 probes
Affymetrix “Gene Chips”Probes:Oligos (25 mer),represent gene fragmentsProduced by photolithography on silicamatrix500,000 probes
Commercially available Oligonucleotide microarrays
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tumor normal
Mixing
SPOTTED MICROARRAYS
Pro
Only one chip needed perexperiment
Con
Absolute gene expression levelscannot be measured
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cDNA
GENECHIPS® BY AFFYMETRIX
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GENECHIPS® BY AFFYMETRIX
Single nucleotide polymorphism (SNP):responsible for genetic variation and thesource of susceptibility to genetically causeddiseases
SNP microarrays:particular type of DNA microarrays used toidentify genetic variation in individuals andacross populations
Applications:ForensicsMeasurement of genetic predispositionto diseaseProfiling somatic mutations in cancer
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MICROARRAY“Heat map”
Here we can see an annotated close-up of an affymetrix chip, with the regionsrelating to several genes highlighted.
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GENECHIPS GENECHIPS®® BY AFFYMETRIX BY AFFYMETRIX
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THE PROBLEM
NEED TO QUANTITATE DIFFERENCES IN mRNA EXPRESSION
THE SOLUTION
• PCR:- most sensitive- can discriminate closely related mRNAs- technically simple- but difficult to get truly quantitative results using conventional
PCR
Real time PCR was developed because of the need to quantitate differences inmRNA expression. PCR methods are particularly valuable when amounts ofRNA are low since the fact that they involve an amplification step means theyare more sensitive.
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RT-PCR REVERSE TRANSCRIPTASE-PCR
• RNA containing virus– PCR doesn’t work on RNA
templates
– RT PCR• make cDNA copy of
RNA sequence first
• PCR the cDNA copy ofRNA
Extract RNA from cells
RNA
PCR
Can observe very low levels of expressionRequires very small amounts of mRNA
Have to design multiple custom primers for each gene
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RT-PCR
Measures relative expression of mRNA
1. Isolate and purify mRNA2. reverse transcription3. PCR amplification4. run on gel
First, the mRNA’s are isolated and purified.Next, the mRNA is reverse transcribed, possibly using a gene-specific primer.Recall that transcription normally makes an RNA copy from a DNA template.This is reverse transcription, as it is making a DNA copy from an RNAtemplate.Next, standard PCR is used to amplify the number of copies of the transcriptunder study.Finally, the resulting product is run out on a gel and probed.
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PCR
Regular PCR involves performing the reaction and electrophoresing thefinal product – not reflective of starting amount
Cycle number0 35
100 ConventionalPCR
Prod
uct A
mou
nt
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WHAT’S WRONG WITH AGAROSE GELS?
* Poor precision* Low sensitivity* Short dynamic range < 2 logs* Low resolution* Non-automated* Size-based discrimination only* Results are not expressed as numbers* Ethidium bromide staining is not very quantitative
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REAL TIME PCR
• kinetic approach• early stages• while still linear
Real-time PCR monitors the fluorescence emitted during thereaction as an indicator of amplicon production at each PCR
cycle (in real time) as opposed to the endpoint detection
Real time PCR is a kinetic approach, where you look at the reaction in theearly stages while it is still linear. There are many real time machinesavailable. This is the one we use (the BioRad Icycler IQ real time PCRinstrument). The lid slides back and then we put samples in a 96-well plateformat inside, so one can look at a lot of samples simultaneously. The machinecontains a sensitive camera which monitors the fluorescence in each well ofthe 96-well plate at frequent intervals during the PCR reaction. In our case, asDNA is synthesized, more SYBR green will bind and the fluorescence willincrease.
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REAL-TIME qPCR
• What is real-time quantitative PCR?
DNA amplification is monitored as the reaction occurs
A PCR-based method to measure the number ofcopies of a particular DNA fragment in a given sample
- Amplification products are labeled by a DNA binding dye orprobe chemistry that emit fluorescent signal when excited.-The signal strength of the emitted light is directly proportional tothe amount of PCR product in the reaction-The fluorescence intensity is detected and recorded every cycle
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REAL-TIME PCR
• Real-Time PCR based on PCR(1983, Kary Mullis)
• PCR thermocycler, UV lamp, camera and EtBr
• EtBr integrated in ds DNA is detected by UV
Advantages Real-Time PCR
• Reproducible DNA- und RNA quantification• No electrophoresis• Large dynamic area (up to 9 Logs)• High throughput
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QUANTITATIVE PCR (qPCR)
Cycle number0 35
100 ConventionalPCR
RealtimeqPCR Pr
oduc
t Am
ount
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REAL-TIME QPCR VS.TRADITIONAL PCR
StartingTemplate
PCRRound 1
PCRRound 2
2XTemplate
4XTemplate
CycleFl
uore
scen
ceReal-time analysisDetection and constantmonitoring of amplificationproducts is possible duringthe entire run. Analysisand quantification can bemade in the logarithmicphase of the reaction ratherthan at the end of thereaction.
30-40 more cycles
End-point analysisAgarose gel for productdetection
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PRINZIP DER TAQMAN-PCR
+ specific - expensive
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SYBR GREEN
• Second method of detection ofPCR product– Detecting an increase in
fluorescence intensity
• Utilize a fluorophore that emitslight only when complexed withdsDNA
• Increasing amount of dsDNAproduct with each round ofamplification gives acorresponding increase influorescence
Fluorescence low
Fluorescence high
+ unexpensive - unspecific
In this presentation, we will be using Sybr green to monitor DNA synthesis.Sybr green is a dye which binds to double stranded DNA but not to single-stranded DNA and is frequently used to monitor the synthesis of DNA duringreal-time PCR reactions. When it is bound to double stranded DNA itfluoresces very brightly (much more brightly than ethidium bromide does,which is why we use Sybr Green rather than ethidium bromide; we also useSybr green because the ratio of fluorescence in the presence of double-strandedDNA to the fluorescence in the presence of single-stranded DNA is muchhigher that the ratio for ethidium bromide). Other methods can also be used todetect the product during real-time PCR, but will not be discussed here.However, many of the principles discussed below apply to any real-time PCRreaction.
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iCYCLER (BIORAD)
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QUANTITATIVE PCR (QPCR)
Cycle number0 35
100
Create a standard curve with 10-fold serial dilutions of PCR product –assign arbitrary values
Compare values from standards with values for unknown sample
Pro
duct
Am
ount
STD 1: 1,000,000STD 2: 100,000STD 3: 10,000STD 4: 1,000STD 5: 100STD 6: 10
Sample: 6,592
Plateau information is not quantitative
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STANDARD DILUTION
Ct value
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REAL-TIME PCR ADVANTAGES
* not influenced by non-specific amplification
* amplification can be monitored real-time
* no post-PCR processing of products(high throughput, low contamination risk)
* ultra-rapid cycling (30 minutes to 2 hours)
* wider dynamic range of up to 1010-fold
* requirement of 1000-fold less RNA than conventional assays(3 picogram = one genome equivalent)
* detection is capable down to a 2-fold change
* confirmation of specific amplification by melting point analysis
* most specific, sensitive and reproducible
* not much more expensive than conventional PCR(except equipment cost)
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PCR BIAS:LIMITATIONS TO QUANTIFICATION
• Some genes may amplify more readily• Some cycles may be more efficient• A reagent may become limiting in the reaction
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REAL-TIME PCR DISADVANTAGES
* setting up requires high technical skill and support
* high equipment cost
* * *
* intra- and inter-assay variation
* Littte RNA stability
* DNA contamination (in mRNA analysis)
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To control the quality of RNA, researcher can use the BioanalyzerAgilent Technologies which is a microfluidic Lab-on-a-Chiptechnology. You can have a qualitative and quantitative analysis ofRNA in 1 hour (one Agilent Chips included 12 RNA Samples).
Capacity optimale : 1 chip/ hour (12 RNA sample)
RNA-QUALITY CONTROL: AGILENT BIOANALYZER(LAB-ON-A-CHIP)
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18S
28S
Flu
ore
scence
Time (seconds)
0
5
10
15
20
25
30
35
40
45
50
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Distinct 28S ribosomalsubunit (or prok. 23S):ideally 2X size of 18S
Distinct 18Sribosomal subunit(or prok. 16S)
Marker
Marker
28S
18S
INTACT TOTAL RNA
~100 bp
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HEAVILY DIGESTED RNA
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COMPLETELY DIGESTED RNA
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S
Flu
ore
scence
Time (seconds)
0
25
50
75
100
125
150
175
19 24 29 34 39 44 49 54 59 64 69
~100 bp
Marker
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A PROPER LADDERF
luore
scence
Time (seconds)
0
5
10
15
20
25
30
19 24 29 34 39 44 49 54 59 64 69
25 bp
200 bp
500 bp
~6 kb
4 kp1 kb
2 kb
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RNA METHODS: RNASE PROTECTION
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RNA METHODS: RNASE PROTECTION
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Pros– Medium through-put (~ 10 genes)– Good reproducibility allow statistical analysis of tests– Cost-effective
Cons– Gel-based– 2-3 days to complete
RNA METHODS: RNASE PROTECTION
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COMPARISON OF QUANTITATIVE ASSAYS
Real-Time PCR
MicroarraysRPA
Northern
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101
102
103
104
105
106
107
108
108
107
106
105
104
103
102
101
100
Dynamic RangeSensitivity
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RNA IN SITU HYBRIDISATION
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