Was given 1ug of each of these two RNA samples and processed them with Promega’s PolyA Tract Kit according to protocol. After elution, the samples were EtOH precipitated @ -20C for 30mins, pelleted 30mins 16,000g for 30mins, 4C. Supe removed, RNA washed with 1mL 70% EtOH and spun 15mins 16,000g, 4C. Supe removed. Resusupended in 15uL of 0.1% DEPC-H2O and spec’d.
Results: No measurable amount of RNA in either sample. Samples were stored @ -80C.
DNased RNA from earlier today was split into four equal parts (175uL = 39.8ug). Three will be used for mRNA isolation and the fourth will remain as total RNA. Three of these were precipitated according to Ambion PolyAPurist Protocol: 1/10 volume 5M ammonium acetate, 1uL glycogen and 2.5 volumes of 100% EtOH. Incubated @ -80C for 30 mins. One sample was processed with the Promega PolyA Tract kit. The remaining two samples were processed according to PolyAPurist Protocol. Of those two, one of the samples was processed a second time to evaluate the effectiveness of running a sample through the PolyAPurist Protocol twice.
mRNA samples were precipitated O/N @ -20C according to the PolyAPurist Protocol.
mRNA was isolated according to Ambion PolyA Purist protocol. After mixing samples with resin, samples were incubated @ RT for 1hr. Samples were washed per the protocol. However, the hemo sample was not clearing from the spin columns with the protocol-directed 3 min. spins. The column had to be spun up to 15 mins. in order for the column to clear.
Results: The gill mRNA looks good (~1.8ug). The concentration of the hemo sample is extremely low and is below the error threshold for the NanoDrop, but it may not be that bad. The samples are in a relatively large volume (~200uL) and the yield is expected to be very small. So, I will precipitate these samples O/N @ -20C according to Ambion PolyA Purist protocol in order to concentrate them.
The two RNA samples from yesterday were precipitated and washed according to the Ambion PolyA Purist protocol and resuspended in 50uL of 0.1% DEPC-H2O.
Results: RNA readings look better than they did prior to precipitation. The hemo RNA samples will be combined with previous hemo RNA samples and mRNA will be isolated using the Ambion PolyA Purist Kit.