Tag Archives: gonad

mRNA Isolation – Herring gonad/ovary RNA (from 20091023)

RNA Precipitation

Sample was spun 16,000g, 30mins, 4C. Supe removed. Pellet washed with 1mL 70% EtOH. Spun 16,000g, 10mins, 4C. Supe removed. Pellet resuspended in 250uL of nuclease free H2O. Will proceed with mRNA isolation.

 

mRNA Isolation

Isolated mRNA using Ambion’s MicroPolyA Purist Kit according to protocol. Performed two rounds of isolation to decrease residual rRNA carryover that we frequently see after a single round.

Results:

Started with ~90ug of total RNA. Yield of mRNA = 3.26ug. That is a ~3.6% recovery of mRNA.

RNA Precipitation – Herring gonad/ovary RNA (from 20091023)

A subset (3 samples from each group) of samples were pooled (see spreadsheet, green-highlighted samples), each providing ~7.5ug of RNA, yielding 112.17uL. 0.1 vols of 3M NaOAC, pH = 5.2 were added to the tube (11.22uL). 2 vols of EtOH (246.8uL) was added to the tube. Tube was vortexed to mix and incubated @ -20C O/N.

RNA Isolation – Herring Gonad/Ovary Samples

RNA was isolated according to protocol. Pellets were resuspended in 50uL of 0.1%DEPC-H2O, heated @ 55C for 5 mins, spec’d and stored @ -80C in the “Herring RNA Box #1″.

Results:

Most of the samples look good, however there are a number of samples that are downright bad. Either no RNA or very low concentrations with poor 260/280, 260/230 ratios.

RNA Isolation – Herring Gonad/Ovary Samples

From the Seeb Lab. Homogenized entire gonad/ovary samples in 5mL of TriReagent with the sonicator. In essence, based on the manufacturer’s recommendation, this means the ratio of tissue:TriReagent was ~2x. Transferred 0.5mL of homogenized gonad/ovary sample to 1.5mL snap cap tubes and added an additional 0.5mL of TriReagent, to adjust the ratio of tissue:TriReagent to ~1x. Samples were then stored @ -80C. These will be further processed tomorrow.

RNA Isolation – Hard clam hemo (from 20090121)

The 8 hemo samples were pooled and the 4 gonad/d.g. samples were pooled. RNA was isolated. The homo sample was resuspended in 100uL of 0.1%DEPC-H2O and the gonad/d.g. sample was resuspended in 50uL 0.1%DEPC-H2O. RNA was precipitated O/N @ -20C according to Ambion PolyA Purist protocol in preparation for mRNA isolation.