Steven asked that I isolate RNA from the remainder of the Berdahl brain tissue samples (GitHub issue).
For reference, here’s the original info provided regarding these samples (GitHub issue).
Sample labelling was as follows:
Numbers refer to individuals:
1-15 are territorial individuals
16-30 are social individuals
Letters refer to tissues:
- brain
- liver
- gonads
The following brain samples were selected for RNA isolation:
- 6A
- 7A
- 8A
- 9A
- 10A
- 11A
- 12A
- 13A
- 14A
- 15A
- 17A
- 18A
- 19A
- 21A
- 22A
- 24A
- 26A
- 27A
- 28A
- 29A
- 30A
RNA was isolated using RNAzol RT (Molecular Research Center) and the Direct-zol RNA Microprep Kit (ZymoResearch), with the DNase I on-column treatment step. The brain tissues were split into 4 equal size portions. Each portion was homogenized individually. Forceps were rinsed in distilled H2O, soaked in 10% bleach solution for 10mins, and then rinsed in distilled H2O before re-use. All centrifugation steps were performed at 16,000g for 1 min. Here’s a brief overview of the process.
Tubes containing tissues were allowed to thaw completely at room temperature (RT). Tissues were homogenized in 500uL of TriReagent with “disposable” platic mortar/pestle tubes (1.5mL). After homogenization, an additional 500uL of TriReagent was added to the tube, vortexed and incubated at RT for 15mins. Homogenized samples were stored over night at -20o.
The four tubes from each sample was pelleted and supernatant was transferred to a 15.0mL tube. An equal volume (4mL) of 100% ethanol was added to this supernatant and mixed thoroughly by vortexing. Direct-zol Microprep Kit protocol was followed from here on, including on-column DNase I treatment. Samples were eluted with 15uL of H2O.
RNA was quantified using the Roberts Lab Qubit 3.0 using the Qubit RNA High Sensitivity assay.
All RNA was stored @ -80oC in Sam’s RNA Box #3.
RESULTS
Raw Qubit data (Google Sheet):
SUMMARY TABLE:
| Sample | Concentration (ng/uL) | Elution Volume (uL) | Yield (ng) |
|---|---|---|---|
| 6A | 116 | 15 | 1740 |
| 7A | 132 | 15 | 1980 |
| 8A | 120 | 15 | 1800 |
| 9A | 130 | 15 | 1950 |
| 10A | 94 | 15 | 1410 |
| 11A | 114 | 15 | 1710 |
| 12A | 80 | 15 | 1200 |
| 13A | 80.8 | 15 | 1212 |
| 14A | 74.6 | 15 | 1119 |
| 15A | 108 | 15 | 1620 |
| 17A | 138 | 15 | 2070 |
| 18A | 89 | 15 | 1335 |
| 19A | 67.6 | 15 | 1014 |
| 21A | 61.6 | 15 | 924 |
| 22A | 108 | 15 | 1620 |
| 24A | 83.8 | 15 | 1257 |
| 26A | 98.8 | 15 | 1482 |
| 27A | 110 | 15 | 1650 |
| 28A | 118 | 15 | 1770 |
| 29A | 124 | 15 | 1860 |
| 30A | 102 | 15 | 1530 |