Reverse Transcription - C.gigas Lifestage Carryover RNA

INTRO

Continuing with Ariana’s request to extract RNA and run qPCRs (GitHub Issue) for the lifestage_carryover project (GitHub repo), I performed reverse transcription on RNA I previously isolated:

• 20240615 and 20240617 (Notebook)
• 20240618 (Notebook)
• 20240701 (Notebook)

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, low profile 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 most reactions, but there were a number of samples which had insufficient RNA to accommodate that amount. In those instances, the maximum volume of RNA which fit into the reaction volume was used (i.e. 9uL of RNA). Those samples, and the actual quantity of RNA used for each, are listed below:

sample	RNA in RT Rxn (ng)
213	43.2
227	43.2
231	166.5
238	171
287	139.5
371	59.04

Briefly, here is how the reactions were setup:

1. Combined RNA (200ng), oligo dT primers, and H₂O to a final volume of 10uL.
2. Incubated plate at 70^oC for 5mins and immediately chilled plate in ice water for 5mins.
3. Added 30uL of RT reaction master mix to each sample.
4. Incubated plate at 25^oC for 5mins.
5. Incubated plate at 42^oC for 5mins.
6. Incubated plate at 70^oC for 15mins to deactivate reverse transcriptase.

Important

Step 6 is required if using cDNA for downstream qPCR analyses.

cDNA calcs and plate layout:

• cgig-lifestage-carryover-cDNA-calcs (Google Sheet)

Plate was stored at -20^oC.