Department of Pharmaceutics
School of Pharmacy
Center for DNA Sequencing and Gene Analysis
H260 Health Sciences Center
Box 357610
Seattle, WA 98195 -7610
(206) 616-8718
Fax: (206) 543-3204
Version 1.0, July 25, 2003
Standard Protocol for Generating cDNA from Total RNA for RealTime™ PCR
1.
Keep RNA on ice at all times and wear gloves at all times to avoid Rnase
contamination. Use barrier tips only to avoid cross-contamination and only use pipettes
devoted to PCR work.
2.
It is recommended that RNA samples be analyzed for integrity of the 28s & 18s
bands by gel electrophoresis or Agilent Bioanalyzer.
cDNA Synthesis Protocol
Each reaction utilizes 1 μg total RNA in a final volume of 10 μL
All of the listed reagents except water are from ABI’s TaqMan ® reverse transcription kit
(catalog # N808-0234)
We prepare an RT master mix containing all components except RNA and water. Each
sample of RNA is mixed with Rnase-free water to a final volume of 3.85 μL and then
6.15 μL of RT master mix is added to RNA in 200 μL PCR tube on ice.
RT Master Mix:
1 μL 10X RT buffer
2 μL dNTP mix (500 μM final for each dNTP)
0.5 μL oligo dT or random hexamers (2.5 μM final)
2.2 μL MgCl2 (5.5 mM final)
0.2 μL Rnase inhibitor (0.4 units final)
0.25 μL Multiscribe RT (1.25 units final)
Place sample tubes in PCR machine and follow this profile:
45°C for 50 minutes, 85°C for 10 minutes then hold at 4°C
Dilute samples with TE to either 50 μL or 100 μL for analyzing either 100 ng or 50 ng by
RealTime® PCR, respectively.
For RealTime® PCR, add 5 μl cDNA to each well of the plate along with 12.5 μL 2X
MasterMix and primers and probe in a final volume of 25 μL
Room H260 Health Sciences Building, Box 357610 Seattle, Washington 98195-7610 (206) 616-8718 FAX: (206) 543-3204
edkelly@u.washington.edu http://depts.washington.edu/pceut/pceut_services/index.html