qPCR – Water Filter cDNA for RLO Viability Assessment

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:

  • T1A
  • T1B
  • T3A
  • T3B

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…



Reverse Transcription – Water Filter DNased RNA (from 20161207)

Performed reverse transcription on the DNased RNA samples that I verified were free of detectable RLO DNA (20161207).

Combined 17μL DNased RNA + 0.5μL random primers (Promega; Cat: C1181) in 0.2mL PCR tubes.

NOTE: The 17μL was virtually all of the sample volume recovered from DNasing. As such, the DNased RNA will not be quantified.

Incubated DNased RNA and primer mix in PTC-200 thermal cycler (MJ Research) at 70C for 5mins w/heated lid, then immediately placed on ice.

Created master mix of following components:

5x MMLV RT BUFFER 5 14 70
10mM dNTPS (Promega) 1.25 14 17.5
MMLV RT (Promega) 0.5 14 7

Added 6.75 of master mix to each and mixed by pipetting.

Incubated PTC-200 thermal cycler (MJ Research) @ 37C for 1hr (no heated lid), followed by 95C for 3mins (heated lid). Samples were transferred to 0.5mL snap cap tubes and labelled with “cDNA” and the corresponding sample name. Samples will be stored in my -20C box.

UPDATE 20170830 Lisa has moved these samples to a -20C box dedicated to RLO Viability cDNA.

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.

qPCR – Withering Syndrome Water Filter cDNA

Ran qPCR on withering syndrome water filter cDNA from yesterday.

Master mix calcs are here: 20150319 – qPCR WSN cDNA

Ran p18RK7 standard curve from 20120731.

All samples were run in duplicate.

Plate layout, cycling params, etc. can be found in the qPCR Report (see Results).

qPCR Report (PDF): Sam_2015-03-19 09-47-31_CC009827.pdf
qPCR Data File (CFX96): Sam_2015-03-19 09-47-31_CC009827.pcrd

Standard curve amplified properly and looks perfect.

No amplification in any other samples.

Will plan on verifying cDNA is actually good by (which I should have thought to include on this run) qPCRing with some universal bacterial primers.


Reverse Transcription – Withering Syndrome DNased RNA

Performed reverse transcription with 307ng of each DNased withering syndrome RNA from earlier today using Promega reagents (M-MLV, random primers) according to the manufacturer’s recommendations in the M-MLV product insert.

Reverse transcription calcs are here: 20150318 – WS Viability cDNA Calcs

Incubated DNased RNA/primer mixture @ 70C for 5mins. Placed immediately on ice. Added M-MLV master mix to each sample, incubated @ 37C 1hr and held at 4C in thermalcycler.

qPCR – Colleen’s Oyster cDNA 2GE with OsHV NGS Primers

A sample that was not in the strip of cDNA tubes was not qPCRd in the last run (20110518). Ran that sample (labelled 2GE R 1/5), as well as positive control DNA (07-CB-719-3), 1GE (cDNA) and 3GE (cDNA). Master mix calcs are here. Plate layout, cycling params, etc. can be found in the qPCR Report (see Results).


qPCR Report (PDF)

Overall, samples look good (no signal in NTCs, clean melt curves, reps are good). Data has been sent to Colleen for analysis.

qPCR – Abalone Virus (Primers AbV03 and AbV06)

Repeated abalone virus qPCR from 20110201 since signals came up in the NTC, all signals came up after 40 cycles, and I didn’t have a melting curve in the protocol. Additionally, added twice the volume of cDNA (4uL instead of 2uL) in hopes of increasing detection. qPCR master mix calcs are here. See the qPCR Report (Results) for cycling params/plate layout/etc.


qPCR Report (PDF)

Essentially no amplification in any samples. Currently, the only issue that I can think of is that this current batch of cDNA was made using only 200ng of RNA. The earliest batch of cDNA that was used when these primers were first tested (DATE!!!) was made with 1000ng of RNA. Unfortunately, we are low on RNA for most of these samples and making more cDNA from 1000ng of RNA is likely not feasible. Will double-check RNA status to see what remains. Additionally, no Dg tissue remains for these samples.

qPCR – Virus Primer Optimization: AbV05

Performed qPCR using cDNA pool from 20110106 that was set up with both an annealing temp gradient as well as varying concentrations of MgCl2. qPCR master mix calcs are here. Plate layout, cycling parma, etc can be found in the qPCR report (see Results below).


qPCR Report (PDF)

Based on the signals in the negative control wells (NTC), it would appear that this primer set won’t work. Potentially significant primer dimers contributing to the signals seen in the NTCs.