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
3
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
RNA isolation
~ 1%
mRNA
tRNA
• 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
Only mRNA has a poly-Adenin tail at the 3’ end
AAAAA
rRNA
Sampling and Template Preparation
 Important to be familiar with general principles of working with RNA:
 Avoid RNAses
 Always 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
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
8
RT–PCR at the bench
RT:
Add:
total RNA + oligodT
65ºC – 10 min
denature
37 ºC – 1 hour
RNasin
dNTPs
RT ready
anneal + elongate
Enzyme
PCR:
template
Buffer
95ºC
3 min
MgCl2
dNTPs
95ºC – 30 sec
55ºC – 30 sec
72ºC – 1 min
72ºC
10 min
30 cycles
denature
amplify
PCR ready
finish
DNA pol
primers
Gel analysis
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
Conventional PCR utilizes two primers and products are
detected by gel electrophoresis
“cPCR”
Agarose gel
electrophoresis following
PCR
Log Target

Cycle Number
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’
∆ Fluorescence

Threshold
CT
CT
Cycle 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
∆ Fluorescence

Greater starting target
Less starting target
CT
<
CT
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
Step 1:
Anneal and
Polymerization
Forward Primer
R
Taq
Polymerase
Probe
Q
Reverse Primer
R
T
Step 2:
Strand Displacement
Q
R
Step 3:
Cleavage
Polymerization Complete
Q
Probe must hybridize specifically for cleavage
A probe is cleaved each time a target is copied
Energy from fluorophore transferred to
quencher
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