Reverse Transcription - R.philippinarum Pooled Gill RNA from 20261027

INTRO

Continuing with the Manila clam (R.philippinarum) RNA which I quantified and pooled yesterday (20261027), today I performed reverse transcription using the GoScript Reverse Transcription Kit (Promega).

MATERIALS & METHODS

I ran the GoScript Reverse Transcription Kit (Promega) according to the manufacturer’s protocol using the pooled Manila clam RNA, but I tripled the reaction volume (20uL -> 60uL) to accommodate the number of qPCR reactions I would need to run with the 13 primer sets, in triplicate, which we’ll be testing.

Protocol

Briefly,

1. RNA denaturation and primer annealing.

• 300ng of total RNA
• oligo dT primers
• final volume = 15uL
• incubated 70^oC 5mins
• put on ice water for at least 5mins

2. Reverse transcription

• final volume 60uL
• incubated 25^oC 5mins
• incubated 42^oC 60mins
• incubated 70^oC 15mins

Important

Final incubation (inactivation) is required for successful downstream qPCR analysis!

Calculations

• 20261028-rphi-gill_pool-cDNA-calcs (Google Sheet)

Resulting cDNA was temporarily stored on ice for use in qPCR reactions and then stored @ -20^oC.