DNAse Treatment
In 200 μ l PCR tube or 96-well plate add RNA and water followed by the DNAse mastermix as shown below.
Total RNA……………………….. up to 5 μ g
ddH₂O ……………………………. volume to 10.2 μ l
DNAse I 10X Buffer ………….1.52 μ l
DNAse I (1 U/ul) ………………3.48 μ l
Total Volume ……………………15.2 μ l
Vortex, spin and incubate at 37 ° C for 20-30 minutes.
Stop reaction, add 1.5 μ l stop buffer or 40 mM EDTA (1ul stop per 10ul DNAse rxn).
Heat kill DNAse enzyme, incubate at 65 ° C for 5 minutes.
Immediately place on ice for 2 minutes, spin quickly and place back on ice.
Denaturation and Annealing of Oligo dT
Add 1 μ l Oligo dT to tube from above to bring volume to 17.7 μ l.
Vortex, spin.
Heat to 65 ° C for 5 minutes.
Place on ice immediately, spin and place back on ice. (Do not let RNA reach room temp after incubation)
1 st Strand Synthesis
(Modified for adding 17.7 μ l from above DNAse treatmentt to make a 30ul rxn for SSIII, for Promega or Invitrogen M-MLV RT, sub H₂O for .1M DTT)
Prepare a master mix per reaction as follows
5X reaction buffer …………………..6 μ l
25 mM dNTP ………………………….8 μ l
Water …………………………………….1.5 μ l
.1M DTT……………………………….. 3 μl
Reverse Transcriptase …………..1 μ l
Total Volume …………………………12.3 μ l
Add 12.3 μ l of master mix to each well/tube.
Incubate at 50 ° C for 60 minutes (for M-MLV, incubate at 37 ° C)
Inactivate the reaction by heating at 70 ° C for 15 minutes.
Extraction of cDNA (optional)
Add 170 μ l water to each tube
Add 1 volume (200ul) Phenol:Chloroform, vortex.
Centrifuge at max speed for 5 minutes.
Transfer aqueous layer to fresh 1.5 ml tube and precipitate cDNA with .1 vol (18 μ l) NaAOC pH 5.3 and 2 volumes (360 μ l) 100% EtOH (can also add a carrier at this point, glycogen, etc to aid in pelleting of cDNA)
Invert several times and inubate on ice for 20 minutes.
Spin at 4 ° C for 10 minutes at max speed.
Pipette off EtOH and wash with 75 % EtOH, spin 2 minutes at max speed.
Pipette of EtOH, being careful to avoid cDNA pellet.
Allow pellet to dry and resuspend in 30 μ l ddH 2 O
RQ1 RNase-Free DNase (Promega #M6101)
Oligo dT 15 Primer (Promega #C1101)
Oligo dT 12-18 Primer (Invitrogen # 18418-012)
SuperScript III Reverse Transcriptase (Invitrogen #18080-044)
M-MLV Reverse Transcriptase (Invitrogen #28025-013)
M-MLV Reverse Transcriptase (Promega #M1705)