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.
Tag Archives: Pacific herring
mRNA Isolation – Herring Liver RNA (from 20091021)
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 ~500ug. Total yield = 5.3ug. That is a 1.06% recovery of mRNA.
RNA Precipitation – Herring Liver Samples
A subset of samples (4 samples from each group) were pooled (see spreadsheet, green-highlighted samples), each providing ~68ug of RNA. The pooled sample was split into two tubes (435uL/tube). 0.1 vols (43.5uL) of 3M NaOAC (pH=5.2) were added, then 2 vols of 100% EtOH (957uL). Tubes were vortexed and incubated @ -80C for 30mins. RNA was pelleted by spinning 16,000g, 30mins, 4C. Supe was removed, pellets washed with 70% EtOH. Tubes were spun 16,000, 10mins, 4C. Supe removed. Pellets were resuspended in 250uL of RNase-free H2O and stored @-80C until Monday for mRNA isolation.
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 – Herring Liver Samples
RNA was isolated according to protocol. Pellets were resuspended in 200uL of 0.1%DEPC-H2O, heated @ 55C for 5 mins, spec’d and stored @ -80C in the “Herring RNA Box #1″.
Results:
RNA looks good. Will speak with Steven how to proceed and whether or not to check for gDNA carryover.
RNA Isolation – Herring Liver Samples (LHPWS09 1-6)
From Seeb Lab. Homogenized entire liver samples in 5mL of TriReagent with the Tissue Tearers. In essence, based on the manufacturer’s recommendation, this means the ratio of tissue:TriReagent was ~2x. Transferred 0.5mL of homogenized liver 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 once all remaining liver samples have been homogenized inTriReagent.