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.
Results:
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.