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.
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.
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)|
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)