mRNA was precipitated according to Ambion’s MicroPolyA Purist Kit protocol. Added 0.1vols of ammonium acetate, 2.5vols of 100% EtOH and incubated 30mins @ -80C. Samples were pelleted, washed with 1mL 70% EtOH, pelleted, resuspended in 8uL of nuclease-free H2O and spec’d:
After precipitation, samples were fragmented with RNase III according to the Ambion Whole Transcriptome Analysis Kit protocol and then cleaned up using the Invitrogen Ribominus Concentration Module, according to the Ambion Whole Transcriptome Analysis Kit protocol. 0.5uL of each sample was removed for analysis on the Bioanalyzer.
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.
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.
Samples from 20091203. 0.5uL was removed from each and transferred to separate tubes and diluted to < 5ng/uL for subsequent Bioanalyzer analysis using the Pico chip. Samples were fragmented using RNase III according to the Ambion WTK protocol and then cleaned up/concentrated using the Invitrogen RiboMinus Concentration Module according to the Ambion WTK protocol.
Samples were spec’d prior to running on the Bioanalyzer:
Concentrations/absorbance values are not accurate when using the NanaDrop after using the RiboMinus Concentration module, according to the Ambion WTK protocol. However, yields seem pretty good…
Total, mRNA and fragmented mRNA from each of the four samples was run on the Pico chip with the Eukaryote Total RNA Bioanalyzer protocol.
The 2L tot (total RNA) and 3L tot (total RNA) samples are clearly very good quality. 2L tot does exhibit some very slight degradation, though. 4L tot (total RNA) and 6L tot (total RNA) show a much greater degree of degradation. All mRNA samples show complete removal of any trace, contaminating rRNA. The fragmented samples (the last four samples on the gel image above) all appear to be perfect. The 4L frag sample simply has less RNA loaded and that is why it is not as dark as the other three fragmented samples. Despite the degradation in the 4L tot and 6L tot samples, the fragmentation profile looks good and we will proceed with making the cDNA libraries for those samples.
EtOH Precipitaiton – Rick’s trout Ribosomoal-depleted RNA for SOLiD WTK (continued from yesterday)
Continued precipitation. Spun samples 30 mins, 16,000g, 4C. Removed supe. Added 1mL 70% EtOH. Spun samples 15mins, 16,000g, 4C. Removed supe. Resuspended in 8uL H2O. Proceeded with SOLiD WTK fragmentation.
Samples were fragmented according to the Whole Transcriptome Kit protocol. Samples were then cleaned up using Invitrogen’s RiboMinus Concentration Module, according to SOLiD WTK protocol. Briefly:
Added 1X volume of binding buffer (100uL)
Added 100% EtOH (250uL)
Eluted with 20uL of H2O.
Samples were spec’d.
Control Sample – Virtually nothing there. Hopefully it’s just too dilute for the NanoDrop, however I have a feeling this sample is bad (degraded?) 1.5uL of the sample has been transferred to a 0.5mL snap cap tube to send off for the Bioanalyzer.
Poly I:C Sample – Looks great, excellent recovery. 0.25uL of this sample was transferred to a 0.5mL snap cap tube containing 1.25uL of H2O to send off for the Bioanalyzer.
Samples were stored @ 80C until resutls from the Bioanalyzer are received.
Prior to starting the procedure, 0.5uL of total RNA was removed from each sample (control, polyI:C), diluted to ~5ng/uL. 1.5uL of each of these was transferred to a 0.5mL snap cap tube for running on the PicoChip on the Bioanalyzer. These were stored @ -80C in the “Bioanalyzer Samples” box.
The remaining total RNA from Rick’s trout RBC (~15uL of the “control” and 20uL of the “polyI:C”) was treated with Invitrogen’s RiboMinus Kit, according to protocol. Samples were then processed following Invitrogen’s Modified RiboMinus Concentration Module, but samples were eluted with 20uL of H2O, instead of 30uL. Samples were spec’d.
The “poly I:C” sample looks good and gave a return of ~800ng, which is ~1% of the total starting RNA (20uL x 0.42ug/uL = 8.4ug). The “control” sample, however, is well short of the expected 1% yield. Recovery was ~220ng, which is only ~0.25% of the total starting RNA (15uL x 0.571ug/uL = 8.565ug). Will proceed to EtOH precipitate the samples in preparation for fragmentation.
Transferred 0.75uL of the “control” sample to a 0.5mL snap cap tube containing 0.75uL of H2O. Transferred 0.25uL of the “poly I:C” sample to a 0.5mL snap cap tube containing 1.25uL of H2O. Samples were stored @ -80C in the “Bioanalyzer Samples” box.
Samples were fragmented according to the Whole Transcriptome Kit protocol. Samples were then cleaned up using Invitrogen’s Modified RiboMinus Concentration Module, according to protocol. Briefly:
Added 1X volume of binding buffer (100uL)
Added equal volume of 100% EtOH so that [EtOH] = 50% (200uL)
Followed remainder of protocol and eluted with 20uL of H2O. Samples were spec’d.
Assuming the NanoDrop readings are accurate (according to the Whole Transcriptome Kit, these may NOT be accurate), got yields of ~93ng for the RBC Control sample and ~132ng for the RBC poly1:C sample. Transferred 1.5uL of each sample to separate 0.5mL tubes for submission to the Bioanalyzer. These were stored @ -80C in the “Bioanalyzer Samples” box. The remainder of the samples were stored @ -80C in the “Samples from Other Researchers” box.