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.
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.
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.
RNA was isolated using 500uL of TriReagent for all samples. Samples were resuspended in 100uL of 0.1%DEPC-H2O and spec’d. Samples stored in Tim’s “NAME OF BOX” box.
Results:
AC# = Air Control Sample
CC# = CO2 Control Sample
AV# = Air Vibrio Sample
CV# = CO2 Vibrio Sample
All the samples look really good, even those that exhibited dark coloration carried over from extraction. Will check for gDNA contamination.
Samples were pelleted at 100g, 4C for 10mins. The supe was mostly removed, leaving ~50uL of supe above the pellets. RNA was isolated according to the TriReagent protocol from the following samples:
3226:
RNAs were resuspended in 10uL of 0.1% DEPC-H2O. They will be DNase treated.
Isolated gDNA according to Molecular Research Center TriReagent protocol from BB#1-20 and DH#1-20. Resuspended DNA in 600uL of 8mM NaOH. Spec.
Results: HORRIBLE! This is some of the worst “DNA” I’ve ever seen. Peaks everywhere EXCEPT at 260nm. Here’s a link to the actual numbers. It’s a text file and is comma separated, so you should open with Excel for it to be readable.
Spoke with Steven. Will pursue RNA instead of continuing down this path for now.
Samples were precipitated according to TriReaget protocol. RNA was resuspended in 50uL of 0.1% DEPC-H2O and then heated @ 55C for 10mins to help dissolve the pellets. Samples will be DNase treated.
Completed the reaminder of the samples (BB#9-20 and DH#1-20) up to the point of precipitation. Isopropanol was added and stored @ -20C. Organic phase was retained for subsequenct gDNA isolation and stored @ 4C.
RNA was isolated from the Control and V.tub+gigas samples from the 0, 1, & 24hr time points using 1mL TriReagent. No visible pellets. Used 20uL of 0.1%DEPC-H2O to resuspend RNA. Incubated @ 55C, 5mins. Spec’d.
Results: RNA looks OK, but not great. For the “V.tub + gigas t=1″ sample, the third spec reading is correct. The first two had the air bubble error.
1ug of RNA in a volume of 12uL was DNAsed using the Ambion DNA-free Kit according to their protocol. RNA was transferred to a fresh tube and stored @ -80C in Sam’s RNA Box #1.
1mL of TriReagent was used to isolate RNA from 3 combined tubes of hemolymph. This resulted in 10 total RNA preps. Pellets were resuspended in 100uL of 0.1% DEPC-H2O and pooled into a single tube and NanoDropped.
Results: RNA solution looked very cloudy and contains a fair amount of insoluble “stuff”. 260/280 ratios also looked bad. Will precipitate O/N according to Ambion PolyA Purist Kit before isolating mRNA tomorrow.