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.
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)