1.Safety precautions
For general safety in laboratory work, consider the general document RWS 004 on environmental safety and work security.
2.Rationale
To synthesize cDNA from RNA samples
3.Application
To be performed by laboratory personnel or persons trained in molecular biology after qualification by an instructor
4.Applicable documents
· RNA Isolation PRBM 001
· GeneAmp 5700 Sequence Detection System PRBI 001
5.Definition
No special
6.Material
· 200 µl-micro reaction tubes
· TaqMan Reverse Transcription Reagents (all from Applied Biosystems, Foster City, CA):
10 x PCR Buffer II (500 mM KCl, 100 mM Tris/HCL, pH 8.3) and
25 mM Magnesium chloride, cat. # N8080010
DeoxyNTPs mixture (2.5 mM each dNTP), cat. # N8080260
Random Hexamer (50 µM), cat. # 8080127
RNase Inhibitor (20 U/µl), cat. # N8080119
MultiScribe Reverse Transcriptase (50 U/µl), cat. # 4311235
· Thermal Cycler 9600 (Applied Biosystems)
· TE (Tris-EDTA) Buffer: prepared from 100x concentrate (SIGMA cat. # T-9285) with DEPC-treated water
7.Procedure
RT reaction mix for 20 µl reaction volume:
The reagents are stored at –20℃. Prior to use, thaw all reagents except the Reverse Transcriptase and the RNase inhibitor. When the reagents are thawed, keep them on ice. Keep also RNA samples and reaction mixture on ice. Keep the Reverse Transcriptase and the RNase inhibitor in the freezer until immediately prior to use.
· Prepare reaction mix (for sample number + 1) by combining all the nonenzymatic components
· Mix the components by pipetting up and down, vortex briefly
· Add the reverse transcriptase and RNase inhibitor
· Mix the components by inverting the microcentrifuge tube
· Dispense appropriate volume (12.3 µl) into 200 µl-micro reaction tubes
· Add RNA sample and DEPC treated water to a reaction volume of 20 µl
· Place samples in Thermal Cycler
· Run 10 min at 25℃ for primer incubation, 30 min at 48°C for reverse transcription, 5 min at 95℃ for reverse transcriptase inactivation (see PRBI 001)
· Optionally dilute the resulting cDNA with TE-buffer and store at –20℃
8.Documentation, Archiving
No special
9. Special Requirements, Quality Assessment
Use gloves and keep tubes closed throughout the procedure. Use aerosol-resistant pipet tips (filter tips).
10. References
TaqMan Gold RT-PCR Kit, Protocol, Applied Biosystems, 1997
