qPCR – Withering Syndrome cDNA Tests

The qPCR on withering syndrome water filter cDNA that I ran earlier today didn’t amplify in any samples, and I neglected to run a positive control primer set on the cDNA to verify that the reverse transcription was successful.

Ran a qPCR using universal 16s primers, EUB A/B.

Additionally, I ran qPCRs using the WSN1 primers on cDNA from black abalone digestive gland (Dg), in case the RNA from the water filters doesn’t actually contain any viable rickettsia-like organisms (RLO).

cDNA templates used:

  • 08:3-7 (from 20090422)
  • 08:3-14 (from 20090422)
  • Day 0-1 (from 20150317)
  • Day 3-1 (from 20150317)
  • Day 7-1 (from 20150317)
  • Day 11-1 (from 20150317)

Note: The black abalone cDNA was made using oligo dT primers, so it’s unlikely to contain many prokaryotic targets.

Withering syndrome positive control:

EUB positive control:

Master mix calcs are here: 20150319 – qPCR WS cDNA test

All samples were run in duplicate. See qPCR Report (see Results) for plate layout, cycling params, etc.

qPCR Report (PDF): Sam_2015-03-19 14-29-09_CC009827.pdf
qPCR Data File (CFX96): Sam_2015-03-19 14-29-09_CC009827.pcrd

WSN1 primers:

There is amplification in the abalone cDNA. This tells us that the withering syndrome qPCR assay will work for detection of cDNA.

No amplification from the water filter cDNA. It suggests that there’s no detectable cDNA in the withering syndrome water filter cDNA .

EUB primers:

There is no amplification in any of the cDNA samples. However, one abalone cDNA produced amplification with the EUB primers, but with an extremely late Cq (Cq = 39) and in only one of the two replicates.

These data suggest that the RNA isolation was unsuccessful. Either the RNA quality is too degraded (we know that the OD 260/280 values are very poor) or there just isn’t sufficient RNA present in the samples to allow us to detect it.