REVERSE TRANSCRIPTION (RT) OF DIATOM RNA USING QIAGEN’S QUANTITECT

Note: RNA should always be diluted in RNAse free water.

1. We generally use 1 µg of total RNA in the reverse transcription reaction.  For each sample, calculate the volume of  the purified RNA solution is required and how much water will be needed to bring the volume to 12 µL.  If the concentration of RNA is low, use 12 µL (and hope for the best). 
2. Thaw RNA samples on ice. Make dilutions if necessary. Use RNase free water for dilutions.
3. Label PCR tubes and add 2 µL of gDNA wipeout buffer to each tube. Place the tubes on ice.
4. Add RNA or RNA+dH₂O [total vol.=12μL] to the tubes containing the  gDNA wipeout buffer.
5. Incubate for 2 min at 42ºC in the PCR machine. Then place tubes immediately on ice.
6. Add 6μL of RT master mix to each of the RNA + gDNA mix. [Final vol. 20μL in each reaction.] Mix by pipetting up and down and store on ice.
7. In the PCR machine, incubate for 30 min at 42ºC then for 3 min at 95ºC to inactivate Quantiscript Reverse Transciptase.  Samples can be held at  4º C until removed from the PCR machine.

Store RT reaction on ice and proceed directly with Real-Time PCR, or for long term storage, store RT reactions at -20ºC.