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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

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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

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105

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107

108

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106

105

104

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102

101

100

Dynamic RangeSensitivity

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RNA IN SITU HYBRIDISATION