ΕΦΑΡΜΟΣΜΕΝΗ ΜΟΡΙΑΚΗ ΒΙΟΛΟΓΙΑ

ΡΑΜΠΙΑΣ ΘΕΟΔΩΡΟΣ

RT-PCR and quantitative RT-PCR for studying gene expression

3

OVERVIEWtissue

extract RNA

copy into cDNA(reverse transciptase)

do real-time PCR or RT-PCR

analyze results

What does the term “RT-PCR” stand for?

Involves two processes:

RT – Reverse Transcription

During this step we synthesize single stranded DNA from RNA template

PCR – Polymerase chain reaction

Using gene-specific primers we amplify a certain part of our gene of interest to get enough amount for further analysis

cDNA synthesis

Let’s start!

total RNA

tRNArRNAmRNA

~ 1%

• Most of the RNA is unimportant for us (tRNA, rRNA)

• mRNA population consists of about 3-5000 different kind

• Strong secondary structure – enzyme cannot work

AAAAAOnly mRNA has a poly-Adenin tail at the 3’ end

RNA isolation

Sampling and Template Preparation Important to be familiar with general principles of working with RNA:

Avoid RNAsesAlways wear gloves when handling reagents or equipment that

will be used in the RNA extraction and reverse transcription procedures

RNAse-free water can be commercially purchased or nanopure water can be treated with diethyl pyrocarbonate (DEPC)

http://www.promega.com/~/media/files/resources/product%20guides/rna%20analysis%20notebook/workingwithrna.ashx?la=en

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IMPORTANCE OF RNA QUALITY• Should be free of protein (absorbance 260nm/280nm)• Should be undegraded (28S/18S ~2:1)• Should be free of DNA (DNAse treat)• Should be free of PCR inhibitors• Purification methods• Clean-up methods

RT–PCR at the bench

total RNA + oligodT37 ºC – 1 hour

anneal + elongate

65ºC – 10 min

denature

Add:

Enzyme

dNTPsRNasin RT ready

RT:

PCR:

DNA pol

dNTPs

primers

Buffer

MgCl2

95ºC3 min

denature amplify

95ºC – 30 sec55ºC – 30 sec72ºC – 1 min

72ºC10 min

finish

PCR ready

template

1-5 ul

Gel analysis

30 cycles

Applications of RT-PCR

• Cloning genes’ expressed forms (not genomic version)

• Monitor a gene’s expression level in any tissue

• Monitor expression changes following treatments

• Sophisticated RT-PCR: The real time PCR

• sequencing a whole mRNA profile

• EST (Expressed Sequence Tags) – database

• Microarray (DNA chip)

• Diagnose and easily differentiate between different cancer types

• Early detection of hidden illnesses

• etc…

Commonly Used PCR Assays

Log

Targ

et

Agarose gel electrophoresis following PCR

Cycle Number

Conventional PCR utilizes two primers and products are detected by gel electrophoresis “cPCR”

http://www.idtdna.com/pages/docs/educational-resources/gel-electrophoresis.pdf

Real Time PCR

Commonly Used PCR Assays Real-time PCR detects a fluorescent signal that is increased

each time a template is copied; the fluorescent signal is monitored each cycle or in ‘real-time’

∆ Fl

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ce

Threshold

CT CTCycle Number

CT = The cycle that a PCR reaction crosses the designated threshold

Also called cycle quantification (CQ) or crossing point (CP)http://www6.appliedbiosystems.com/support/tutorials/pdf/rtpcr_vs_tradpcr.pdf

Commonly Used PCR Assays Quantitative PCR relies on the principal that the quantity of

target at the start of the reaction is proportional to amount of product produced during the exponential phase

∆ Fl

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CT CT

Greater starting target

Less starting target

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Quantitative PCR – in depth• Major assay types

• Fluorogenic 5’ Nuclease Assay• Basis of TaqMan® chemistry• Uses two primers and an internal hydrolysis probe• Most commonly used for fish health diagnostics

• SYBR ® green dye chemistry• Increased fluorescence when bound to dsDNA• Slightly lower specificity• Costs less• May not be as sensitive as the 5’ nuclease assays

http://www.clinical-virology.org/pdfs/PCR_experience.pdf

Quantitative PCR – in depth• Fluorogenic 5’ Nuclease Assay

Forward Primer

Reverse Primer

Step 1:Anneal and

Polymerization

R QEnergy from fluorophore transferred to quencher

RQStep 2:

Strand DisplacementT

RQStep 3:

CleavagePolymerization Complete

Probe must hybridize specifically for cleavageA probe is cleaved each time a target is copied

Probe

TaqPolymerase

Quantitative PCR – in depth• Dual-labeled internal hydrolysis probes

• 5’ reporter dye (typically Fam/Vic etc.)• 3’ quencher (typically non-fluorescent) • Can order from a range of oligo companies• Many companies have proprietary modifications for internal hydrolysis

probes• Minor Grove Binding (MGB) – Applied Biosystems Inc.

• The MGB linker raises the melting temperature of the internal hydrolysis probe and increases probe specificity

http://www3.appliedbiosystems.com/cms/groups/mcb_support/documents/generaldocuments/cms_083618.pdf