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ΕΦΑΡΜΟΣΜΕΝΗ ΜΟΡΙΑΚΗ ΒΙΟΛΟΓΙΑ ΡΑΜΠΙΑΣ ΘΕΟΔΩΡΟΣ. RT-PCR and quantitative RT-PCR for studying gene expression . OVERVIEW. tissue. extract RNA. copy into cDNA (reverse transciptase). do real-time PCR or RT-PCR. analyze results. PCR – Polymerase chain reaction. - PowerPoint PPT Presentation
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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
uore
scen
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
uore
scen
ce
CT CT
Greater starting target
Less starting target
<
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