Isolated RNA in 1mL of Tri-Reagent according to manufacturer’s protocol. Samples were precipitated with isopropanol and stored over the weekend @ -20C. Will conclude isolation on Monday. The samples isolated were:
MA 1-11
MA Vt 1-11
Tag Archives: RNA isolation
RNA Isolation – Various C.gigas Tissue from 20110111
RNA was isolated in 1mL TriReagent, according to protocol. Samples were resuspended in 50uL 0.1% DEPC-H2O and spec’d. RNA was stored @ -80C in “Shellfish RNA Box #4
Results:
RNA looks OK. Not surprising, but mantle and Dg/Gonad tissues ended up with poor OD260/230 ratios. This has been observed in the past with these tissue types.
RNA Isolation – Various C.gigas Tissue from 20110111
RNA was isolated in 1mL TriReagent, according to protocol. Samples were resuspended in 50uL 0.1% DEPC-H2O and spec’d. RNA was stored @ -80C in “Shellfish RNA Box #4“.
Results:
Overall, all RNA looks very good (based on 260/280 and 260/230 values).
RNA Isolation – Various C.gigas Tissue from 20110111
RNA was isolated in 1mL TriReagent, according to protocol. Samples were resuspended in 50uL 0.1% DEPC-H2O and spec’d. RNA was stored @ -80C in “Shellfish RNA Box #4“.
Results:
All gill RNA looks nearly perfect (based on 260/280 and 260/230 values). Muscle RNA is only OK (based on 260/280 and 260/230 values).
RNA Isolation – Hard Clam Tissues Rec’d from Rutgers on 20100820
RNA was isolated from the following samples using TriReagent, according to protocol:
MAX 1, 2, & 3
CA 1, 2, & 3
MA 1, 2, & 3
Samples were resuspended in 50uL of 0.1% DEPC-H2O and spec’d:
Results:
260/280 ratios are decent for most of the samples, with MAX1 and MAX 2 being the exception. Both of these samples also have very poor 260/230 ratios. Out of curiosity, I will EtOH precipitate all samples to see if I can improve both the 260/280 and 260/230 ratios.
RNA Isolation – Sepia samples
Isolated RNA from 7 sepia samples received 20091125. Samples were removed from RNA Later, blotted and homogenized in 500uL of TriReagent. 500uL of additional TriReagent was added to the tubes after homogenization. Procedure was followed normally. The sepia retina RNA was isolated separately from the other samples and was resuspended in 100uL of 0.1% DEPC-H2O. The remaining samples were isolated and resuspended in 20uL of 0.1% DEPC-H2O. Nearly all samples had some sort of purple tint to them, ranging from almost black to extremely faint purple hue. The samples were spec’d and then stored @ -80C in Sam’s RNA Box #1.
Results:
Nearly all of the samples exhibited very strange curves and mediocre 260/280 ratios (for RNA). Could be due to the “purple stuff” carryover or possibly an effect of the RNA Later from which the samples were stored.
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.