Fragmented mRNA according to Ambion’s Whole Transcriptome Sequencing Kit. Cleaned up sample using Ribominus Concentration Module (Invitrogen) according to Ambion’s WTS Analysis Kit. Samples were eluted w/20uL of H2O and stored @ -80C. Will Bioanalyze and speedvac at a later date.
Total and mRNA aliquots (~5ng/uL) were run on the Agilent Bioanalyzer Pico RNA chips.
The gel below shows the comparison/results of total RNA and subsequent mRNA isolations. The gel indicates the following:
- The HPWS09 total RNA (Herring) is totally degraded, but shows the expected profile in the mRNA prep. It would be extremely interesting to see if the degradation has any effect on sequencing, as the mRNA will get fragmented any way in the next step of library construction.
mRNA isolations worked for all samples. Although one might be inclined to say that mRNA isolation did NOT work for the WB sample, one has to take in to consideration that the gel software adjusts the gel contrast to enhance low signals. That’s why all the mRNA samples exhibit a dark background. mRNA generates a broad, relatively weak signal when compared to a total RNA sample. So, the software attempts to boost the low signal for display purposes. Thus, if we were to decrease this signal boosting (or contrast) for the WB mRNA so that the background color matched the WB total RNA background color (white), the rRNA bands visible in the WB mRNA sample would fade to a point where they would not be visible. See the electropherogram overlay (below the gel) for a more visual comparison of this concept.
Electropherogram Overlays of WB total RNA and WB rRNA
The WB total RNA is the red graph which shows extremely high levels of rRNA (as expected). After subsequent mRNA isolation (the blue graph), the rRNA is virtually gone and no longer comprises a significant portion of the sample.
mRNA was pelleted and washed according to Ambion’s MicroPolyA Purist Kit. Pellets were resuspended in 8uL nuclease-free H2O and spec’d. 0.5uL was taken from each sample, transferred to a fresh tube, diluted to ~5ng/uL and stored @ -80C for eventual Bioanalyzer analysis. mRNA samples were stored @ -80C until we receive the Ribominus Concentration Module Kit from Invitrogen (turns out we didn’t have any!) for cleaning up the RNA after fragmentation.
Overall, this mRNA doesn’t look that great. However, I did notice that all samples had (to varying degrees) particulate matter that wouldn’t dissolve. Prior to spec’ing, the particulate matter was pelleted so as to not interfere. All samples will continue to be prepped for SOLiD analysis despite poor 260/280 ratios and low yields.
Received pooled lean and siscowet RNA from Rick. Samples will be processed immediately for SOLiD fragment libraries. Two 1.5mL snap cap tubes labelled:
L.T. 2ug muscle sisco pool
L.T. 2ug muscle lean pool
RNA was first precipitated according to the Ambion MicroPolyA Purist Kit protocol (0.1 vol 5M ammonium acetate, 1uL glycogen, 2.5 vols 100% EtOH). Samples were incubated @ -80C for 30mins. Samples were resusupended in 250uL nuclease-free H2O and spec’d.
Starting quantities PRIOR to total RNA precipitation:
WB tRNA (WB tRNA ~12ug 24.5uL)
PQ tRNA (PW tRNA ~12ug 21.88uL)
CT tRNA (CT tRNA ~12ug 25uL)
1 G/O HPWS09 (20ug)
L.T. 20ug muscle sisco pool
L.T. 20ug muscle lean pool
Removed 1uL of each sample, diluted to ~5ng/uL and stored @ -80C to run on the Bioanalyzer.
Used Ambion MicroPolyA Purist Kit according to protocol. Samples were treated twice to ensure elimination of rRNA from the samples. After second run through MicroPolyA Purist, samples were EtOH precipitated O/N @ -20C according to Ambion’s MicroPolyA Purist protocol.