INTRO
Took me a while to get around to this, but I performed reverse transcription on the remainder of Valentaina’s RNA that I isolated on 20250902. Needed to get supplies and figure out a time to actually process these 94 samples. See the cDNA calcs Google Sheet below for a list of samples.
MATERIALS & METHODS
Reverse transcription was performed using the GoScript Reverse Transcription System (Promega) (PDF), using oligo dT primers and 1.5mM MgCl2. The reaction size was doubled, from 20uL to 40uL, to allow for sufficient cDNA for all the downstream qPCRs we plan to perform. The optional RNasin was also used. Reactions were set up in a 96-well, standard qPCR plate. Plate was sealed with strip caps. The reverse transcriptase components were prepared as a master mix and then distributed to RNA/oligo dT mixtures.
I used 200ng of RNA in the reactions.
Briefly, here is how the reactions were setup:
- Combined RNA (200ng), oligo dT primers, and H2O to a final volume of 10uL.
- Incubated plate at 70oC for 5mins and immediately chilled plate in ice water for 5mins.
- Added 30uL of RT reaction master mix to each sample.
- Incubated plate at 25oC for 5mins.
- Incubated plate at 42oC for 60mins.
- Incubated plate at 70oC for 15mins to deactivate reverse transcriptase.
All incubations were conducted in a 96-well PTC-200 (MJ Research) thermalcycler using a heated lid.
Step 6 is required if using cDNA for downstream qPCR analyses.
cDNA calcs and plate layout:
- 20251019-cgig-cDNA-calcs-polyIC (Google Sheet)
Plate was stored at -20oC.