First-Strand cDNA Synthesis
1. Spin down 10 µg RNA in the Speed Vac until its volume is 15 μL.
(If you spin too long, then bring to volume with RNase free ddH20.)
2. Degrade DNA contamination
Add the following: 1 μL DNAse I (Roche)
4 μL 5X RT Buffer (SS III Kit)
Incubate 37ºC for 30 min.
Heat-kill DNase 70ºC for 15 min.
Place on ice for 5 min.
Centrifuge to collect sample at bottom of tube.
3. Denature nucleotides
Add the following per reaction:
2 μL 10mM dNTP mix
Primer, bring to 8 μL with RNAse-free water
2 μL Oligo(dT)20 (50 μM) OR
400 ng - 1 μg of Oligo(dT)12-18 OR
8 μL Gene-specific primer, 50 µM OR
100 - 500 ng of random hexamers
Denature at 65ºC for 5 min.
Set on ice for at least 1 min.
Centrifuge briefly.
4. Perform RT reaction
Add the following per reaction:
4 μL 5X RT Buffer (SS III Kit)
2 μL 0.1 M DTT
2 μL RNaseOUT™ RNase inhibitor, 40 U/μL
Split the RNA samples equally by pipetting 19 μL to a new tube.
(One will be your - RT control.)
Add 1 μL SuperScript™ III RT (200 U/μL) to each + RT reaction.
(For random hexamers, incubate 25ºC for 5 min.)
Incubate 50ºC for 30-60 min. (For gene-specific primer use 55ºC).
Heat kill at 70ºC for 15 min.
5. Degrade RNA
Add 1 μL RNase H to each tube.
Incubate 37ºC for 20 min.
JL Umphress
10/07/2004
First-Strand cDNA Synthesis (no -RT control)
6. Spin down 5 µg RNA in the Speed Vac until its volume is 7 μL.
(If you spin too long, then bring to volume with RNase free ddH20.)
7. Degrade DNA contamination
Add the following: 1 μL DNAse I (Roche)
2 μL 5X RT Buffer (SS III Kit)
Incubate 37ºC for 30 min.
Heat-kill DNase 70ºC for 15 min.
Place on ice for 5 min.
Centrifuge to collect sample at bottom of tube.
8. Denature nucleotides
Add the following per reaction:
1 μL 10mM dNTP mix
Primer, bring to 4 μL with RNAse-free water
1 μL Oligo(dT)20 (50 μM) OR
200 ng - 500 ng of Oligo(dT)12-18 OR
4 μL Gene-specific primer, 50 µM OR
50 - 250 ng of random hexamers
Denature at 65ºC for 5 min.
Set on ice for at least 1 min.
Centrifuge briefly.
9. Perform RT reaction
Add the following per reaction:
2 μL 5X RT Buffer (SS III Kit)
1 μL 0.1 M DTT
1 μL RNaseOUT™ RNase inhibitor, 40 U/μL
1 μL SuperScript™ III RT (200 U/μL)
(For random hexamers, incubate 25ºC for 5 min.)
Incubate 50ºC for 30-60 min. (For gene-specific primer use 55ºC).
Heat kill at 70ºC for 15 min.
10. Degrade RNA
Add 1 μL RNase H to each tube.
Incubate 37ºC for 20 min.
JL Umphress
10/07/2004