DNase Treatment – Abalone Water Filters for RLO Viability

The RNA I isolated earlier today was subjected to DNase treatment using the Turbo DNA-free Kit (Invitrogen), following the manufacturer’s standard protocol.

After DNase inactivation treatment, the RNA was transferred (recovered ~19uL from each samples)  to a clear, low-profile PCR plate.

The plate layout is here (Google Sheet): 20170309_RLO_viability_DNased_RNA_plate_layout

The samples will be subjected to qPCR to assess the presence/absence of residual gDNA. The plate of DNased RNA was stored @ -80C in the original box that the water filters were stored in.

An overview of the experiment and the various treatments are viewable in the “Viability Trial 2″ tab of Lisa’s spreadsheet (Google Sheet): RLO Viability & ID50

RNA Isolation – Abalone Water Filters for RLO Viability

Water filters stored at -80C in ~1mL of RNAzol RT were provided by Lisa. This is part of an experiment (and Capstone project) to assess RLO viability outside of the host.

The samples were thawed and briefly homogenized (as best I could) with a disposable plastic pestle. The samples were then processed according to the manufacturer’s protocol for total RNA isolation. Samples were resuspended in 25μL of nuclease-free water (Promega).

Immediately proceeded to DNase treatment.

The experimental samples and the various treatments are viewable in the “Viability Trial 2″ tab of Lisa’s spreadsheet (Google Sheet): RLO Viability & ID50

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.

Results:
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…

 

 

RNA Isolation – Abalone Water Filters

Lisa provided me with a set of water filters stored in 1mL RNAzol RT @ -80C to isolate RNA from. This is an attempt to assess withering syndrome viability from within the water.

The samples were thawed and briefly homogenized (as best I could) with a disposable plastic pestle. The samples were then processed according to the manufacturer’s protocol for total RNA isolation. Samples were resuspended in 20μL of 0.1%-DEPC H2O.

Samples were stored @ -80C. Will DNase next week.

The sample names are as follows (the ‘C’ is short for “Control”, per Lisa):

  • T0A
  • T0B
  • T1A
  • T1A C
  • T1B C
  • T3A
  • T3B
  • T7A
  • T7B
  • T7A C
  • T7B C

 

DNA Extraction & Quantification- Ava Withering Syndrome Transmission Study Water Filters

DNA was extracted from filters using the Qiagen DNeasy Blood & Tissue Kit (spin column protocol). Filters were incubated in 400uL of Buffer AL (twice the volume in the Qiagen protocol in order to completely coat the filters) and 50uL of Proteinase K (twice the volume in the Qiagen protocol) at 56C O/N. After incubation, 400uL (twice the volume in the Qiagen protocol) of 100% EtOH was added to each tube and vortexed thoroughly. The supernatant was transferred to the Qiagen spin columns and the Qiagen protocol was followed. Samples were eluted with 100uL of Buffer AE.

After extraction, the samples were quantified using the Roberts Lab Qubit 3.0 (Life Technologies) using the Qubit dsDNA HS reagents. Used 1μL of each sample.

The list of samples are listed below.

Results:

Concentrations are very low for both samples. This may, or may not, be expected, depending on volume of water filtered, where it was collected from, etc.

Raw Qubit Readout (Google Sheet): 20160818_DNA_quant_Qubit_Ava_abalone_WS
Master spreadsheet for these, and future, samples for this project (Google Sheet): Ava WS Transmission DNA Extractions

qPCR – 2011 Ab Endo Water Filters

Ran qPCRs on remaining 2011 water filter DNA samples. All samples were from The Cultured Abalone farm, but some samples are abbreviated “CAB”, while others “TCA”. Samples were extracted 20131001 and 20131120.

Master mix calcs: 20150702 – qPCR WS Ab Endo H2O Filters

All samples were run in duplicate. See qPCR Report in Results below for cycling params.

Standard curve was p18RK7 from 20120731.

Baseline threshold was set to 580, as determined by Lisa.

Results:
qPCR Report (PDF): Sam_2015-07-02 09-45-05_CC009827.pdf
qPCR Data File (CFX96): Sam_2015-07-02 09-45-05_CC009827.pcrd

Data entered in the Ab Endo Samples Google Sheet.

Samples for Abalone Withering Syndrome ddPCR

I selected the following samples (Ab Endo 2011 Water Filter DNA samples) to send to Alice Nguyen at the Marine Science Institute for digital droplet PCR (ddPCR):

  1. CI SRI CP 1A (0 copies)
  2. CI SRI CP 2B (highest)
  3. CI SRI CP 2A (0 copies)
  4. CI SRI CP 1B (high)
  5. CARMEL +500M 1 (medium)
  6. CARMEL +500M 2 (low)

10μL of each sample was sent. Tubes were labelled with “DNA #”. The ‘#’ corresponds to the number in the list above.

10μL of each of p18RK7 qPCR standards (from 20120730) were sent.

Two sets of WSN1 F/R working stocks (10μM) were also sent.

Also sent the QX200 Droplet Generation Oil for EvaGreen Kit (BioRad) that we ordered.

Shipment was on “wet” ice.

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.

Results:
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).

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.

 

qPCR – DNased Withering Syndrome RNA

Ran qPCR on DNased withering syndrome RNA to verify there’s no residual gDNA, prior to performing reverse transciption.

Master mix calcs are here: 20150318 – qPCR WSN DNased RNA Test

Used 1μL of template. This was based on using 17.5μL of DNased RNA in the reverse transcription reaction. Each sample was ~20ng/μL which results in ~350ng DNased RNA in a 25μL reverse transcription reaction (350ng/25μL = 14ng/μL). So, using 1μL in the qPCR reaction roughly approximates the amount of DNased RNA one will encounter when running qPCRs with the cDNA.

Positive control was the 3e6 p18RK7 standard curve sample from 20120731.

All samples were run in duplicate.

See qPCR Report (see Results) for plate layout, cycling params, etc.

Results:

qPCR Report (PDF): Sam_2015-03-18 14-22-39_CC009827.pdf
qPCR Data File (CFX96): Sam_2015-03-18 14-22-39_CC009827.pcrd

The only reaction that amplified was the positive control. The DNased RNA does not have any amplifiable gDNA. Will proceed to reverse transcription.