RNA Isolation and Quantification - Geoduck hemolymph and hemocyte samples

Shelly asked me to isolate RNA and run some qPCRs on the following samples:

-80C Location(rack column row) Sample Name Tissue Reproductive Stage Sex
8 1 3 G-57 H hemolymph(~1mL cells + lymph) 7 F
8 1 3 G-61 H hemolymph(~1mL cells + lymph) 7 F
8 1 1 G-39 H hemolymph(~1mL cells + lymph) 5 F
8 1 1 G-31 H hemolymph(~1mL cells + lymph) 4 F
5 3 1 11/01/2018_1H pelleted hemocytes unknown unknown
5 3 1 11/01/2018_2H pelleted hemocytes unknown unknown
5 3 1 11/8/2018_1H pelleted hemocytes unknown unknown
5 3 1 11/8/2018_2H pelleted hemocytes unknown unknown
5 3 1 11/15/2018_1H pelleted hemocytes unknown unknown
5 3 1 11/15/2018_2H pelleted hemocytes unknown unknown

I was unable to find the 11/01 and the 11/15 samples.

Hemo samples used for RNA isolation in tube rack


RNA was isolated using the Quick-DNA/RNA Microprep Plus Kit (ZymoResearch) according to the manufacturer’s protocol, with the following notes/changes:

  • Added four volumes of lysis buffer for the hemolymph samples

  • Performed on-column DNase step

  • Elution volume = 15uL

After processing, samples were quantified using the Qubit hsRNA Assay, using 2uL of sample. Due to high sample concentrations (i.e. >100ng/uL) I had to dilute the samples a couple of times and re-measure.


RESULTS

Here are the three sets of raw data (Google Sheets):

Here’s the summary table of sample concentrations:

Sample ID Original sample conc. (ng/uL) Sample Vol (uL) Yield (ng)
31 H 25 1000 25000
39H 79 100 7900
57H 20 100 2000
61H 47.7 100 4770
11-08 1H 30.9 15 463.5
11-08 2H 12.3 100 1230

Will do some calculations and performs reverse transcription on these tomorrow, followed by qPCR with vitellogenin primers.

RNA was stored in the -80oC:

  • [Shellfish RNA Box #7: E5 - E9](https://docs.google.com/spreadsheets/d/1ax6C-muxUTXxFEtfWdswBvueLhmxZzmwZcO2ur-0q-Q/edit#gid=1549162576”, “Shellfish RNA Box #7)