Ran qPCRs on the cDNA I made earlier today to determine if there’s any detectable RNA in any of these water filter samples.
Master mix calcs (Google Sheet): 20161208- qPCR WSN1
All samples were run in duplicate. Plate layout, cycling params, etc. are in the qPCR Report (see Results below).
Standard curve was the p18RK7 curve made on 20161128.
Baseline threshold was manually set to 580, as previously determined by Lisa for this assay.
qPCR Report (PDF): Sam_2016-12-08 09-14-38_CC009827_cDNA_WSN1.pdf
qPCR File (CFX96): Sam_2016-12-08 09-14-38_CC009827_cDNA_WSN1.pcrd
Original qPCR File (CFX96): Sam_2016-12-08 09-14-38_CC009827.pcrd
Standard curve looks good.
The following cDNA samples had detectable amplification:
I believe that the labelling scheme represents T1 = Day 1 in water, T3 = Day 3 in water.
These results suggest that the RLO is viable outside of the abalone host for at least three days, but not >= 7 days, although the values are below the theoretical qPCR limit of detection. These results will likely be used to help Lisa with experimental design for a more involved assessment of RLO viability in the water column.
I’ve added the data to Lisa’s spreadsheet (Google Sheet: RLO viability) in the “Expt 1″ worksheet.
Update after talking to Lisa: The water was shipped from a California abalone farm O/N, so T0 = 24hr water. The Control water samples were sea water from our basement facility, not from California.
The fact that there is no amplification at T0 is a bit surprising and possibly suggests that RLO viability outside of the host is on the magnitude of hours, not days…