RT-PCR Protocol
1. Reverse Transcription
For each reaction:
You will need 0.5 to 1.0 μg total RNA
5X RT buffer
10mM dNTPs
100 μM random hexamers
RNase Inhibitor
MMLV RT
Water
RNA
Total
4 μl
1.5 μl
1.0 μl
0.5 μl
1.0 μl
7 μl
5 μl
20
Make up a master mix for N+1 reactions and aliquot 15 μl per tube
Add 5 μl RNA
Perform reaction in PCR machine:
10 min @ 25o
60 min @ 37o
5 min @ 95o
4o Hold
2. QUANTITATIVE PCR
Titanium Taq is from Clontech
We use Titanium Taq for everything except human GAPDH (for some odd
reason the human GAPDH primers only work in the Platinum Taq buffer)
All reactions are done in duplicate.
1. Thaw all reagents at room temperature and then keep on ice.
Thaw cDNA samples and place on ice. cDNA samples can be usually be diluted
1:4 with TE.
2. For each gene, generate a Master Mix which contains everything except
the cDNA sample. Keep on ice.
3. Add 2 µl cDNA sample to each well of the PCR plate or strip.
4. Aliquot 23 µl of Master Mix into each well of the PCR plate or strip.
5. Cover and quick spin.
6. Put into Opticon and start the appropriate program.
For most of the primers that we have developed the following conditions
work well:
QUANTITATIVE PCR WORKSHEET
Titanium Taq
Primer pairs : ________________________
cDNA source :________________________
Amount of cDNA added :___________
Program:_____________________________
Number of Samples :_____
N Reactions= Number of samples + 1
1X
Water
10X PCR Buffer
50 mM MgCl2
10 mM dNTPs
10 µM Primer (F)
10 µM Primer (R)
100X SYBR green
Titanium Taq
Total
16.45
2.5
0.5
1.0
1.0
1.0
0.25
0.3
23
For N reactions
Other Notes:
1. Calculations:
Everything is normalized to the expression of HPRT so the first gene
we always do PCR for is HPRT. Once you have that data, you can go back at
anytime to your RT reaction and test your gene of interest.
Gene expression levels are expressed as relative mRNA units according to
the formula 2(HPRT C(T) – Target C(T)) C(T)= Average threshold cycle of the
duplicate reactions as determined using the Opticon Monitor software.
2. Reagents and sources:
M-MLV reverse transcriptase: Invitrogen # 28025-013
RNase Inhibitor: Invitrogen # 15518-012
Random hexamers: Invitrogen # 48190-011
Titanium taq: BD Biosciences (Clontech) # 639209
Sybr Green: Molecular Probes # S7563
3. Primer design
We design our primers so that they are in separate exons using the
Primer3 program http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi
Tm for the oligos are set to be between 64o and 68o with a product length of
100-200 bps. Optimal size of primers is 20 bases.
4. Reference:
A good source for info on quantitative PCR is from the ABI 7700 User
Bulletin #2.