Ran withering syndrome qPCR on the following RNA samples provided by Lisa to determine if it’s necessary to DNase treat them prior to performing reverse transcription:
I believe these were extracted from water filters collected from basement abalone tank water. The OD260/280 ratios are pretty bad, particularly for RNA, but will proceed anyway.
Master mix calcs are here: 20150318 – qPCR WSN RNA Test
Since I’m anticipating using 1000ng of RNA in the reverse transcription reactions (25μL reaction volume = 40ng/μL), I only used 1μL of RNA (~100ng), and positive control, template in each reaction to semi-accurately represent the quantity of RNA that would be loaded in a qPCR reaction with 2μL of cDNA.
All samples were run in duplicate. Positive control was the 3e6 p18RK7 standard curve sample from 20120731.
All samples are positive for withering syndrome. Will proceed with DNase treatment.