qPCR - Dave’s Manila Calm (Venerupis philippinarum) DNased RNA from yesterday and 20120302

Performed qPCR on all DNased RNA samples from this group (samples #1-48) using beta actin primers (SR IDs: 1379, 1380). 0.5uL of each DNased RNA was used, which was the equivalent of ~40ng, in order to simulate the amount of RNA present in the subsequent cDNA (1000ng of RNA in 25uL cDNA; use 1uL of cDNA in qPCR reaction). Master mix calcs are here. Plate layout, cycling params, etc., can be found in the qPCR Report (see Results). 0.5uL of total RNA from sample Vp gill 01 was used to serve as a positive control, since Dave has no existing V. phillippinarum cDNA.


qPCR Data File (CFX96)

qPCR Report (PDF)

All samples are clean and are ready for reverse transcription.

Of note, the overall fluorescence of the reactions was very low. As such, the default baseline analysis setting (linear) suggested that all samples had a Cq value because the baseline was incorrectly set an was NOT above background fluorescence levels. Changing the baseline analysis setting to “regression” resolved this. Also, it should be noted that one sample (#48) other than the positive control actually does show amplification and a corresponding melt curve. However, the melt curve peak is at a different temp than the positive control, suggesting that this is non-specific amplification in sample #48.