RNA of 8 samples from each group was pooled equally from each individual. RNA was precipitated according to Ambion’s MicroPolyA Purist Kit. Used 0.1 volumes of 3M NaAOc, pH=5.2, 2.5vols of 100% EtOH and incubated 30min @ -80C. Pelleted RNA 16,000g, 30mins. Washed pellet w/70% EtOH and pelleted RNA 16,000g, 15mins. Pellets were resuspended in 50uL nuclease-free H2O and spec’d:
Total RNA pools look really nice. ~45ug of total RNA in each sample.
Isolated mRNA from each pool using Ambion’s MicroPolyA Purist Kit according to protocol. Samples were processed 2x as recommended by Ambion’s SOLiD Whole Transcriptome Analysis Kit. Final elution was 200uL of The RNA Storage Solution. Samples were spec’d: