Tag Archives: WSN1

qPCR – LCM DNA

Ran three primer sets on laser capture microscopy (LCM) DNA samples from 2005 and 2007. Ran the following primer sets:

  • WSN1 (detects RLO)
  • RLOv_helicase (detects RLO phage)
  • XenoCal_prophage

The DNA samples were provided to me by Lisa. I’m not entirely sure of their history:

 

Master mix calcs (Google Sheets):

All samples were run in duplicate. Plate layout, cycling params, etc. are in the qPCR Reports (see Results below).

Standard curves:

Baseline threshold was manually set to 580 for the WSN1 samples, as previously determined by Lisa for this assay.

Baseline threshold was manually set to 580.5 for the RLOv DNA helicase samples, as previously determined by me on 20160128.

 

Results:

WSN1:

 

RLOv DNA helicase:

 

XenoCal prophage:

 

Summary table of all three genes in each sample. Unfortunately, I don’t fully understand the sample name nomenclature, so I can’t really come to any conclusions about the data. Will pass along to Carolyn, Lisa, and Stan.

It’s also important to note that, due to low sample volume, I did not quantify these samples. This is important because any samples listed below that are negative for all three genes can not be conclusively declared “negative”, since we can’t rule out the possibility that they simply lack any DNA.

Presumably they were quantified after their initial extraction?

SAMPLE WSN1 RLOv DNA HELICASE XC PROPHAGE
LCM New RLO 09 + + +
LCM ST RLO 09 - - -
LCM New 08:30-5 B + + +
LCM New 08:30-5 - - -
LCM ST 08:30-3 - - -
LCM WS RLO + - +

 

STANDARD, AMPLIFICATION, & MELT CURVES

 

WSN1

 

 


 

 

RLOv_DNA_helicase

 


 

 

XenoCal prophage

Share

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…

 

 

Share

qPCR – DNased RNA from Abalone Water Filters (from earlier today)

Prior to creating cDNA, need to verify that the DNased RNA from earlier today doesn’t contain any detectable RLO DNA.

Master mix calcs (Google Sheet): 20161128 – qPCR Water Filter DNased RNA

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_2016-12-07 09-10-07_CC009827.pdf
qPCR File (CFX96):Sam_2016-12-07 09-10-07_CC009827.pcrd

Standard curve looks good.

No samples amplified. This suggests that there is no detectable DNA in any of the DNased RNA samples. Will proceed with making cDNA.

 

 

Share

qPCR – Check New Withering Syndrome p18RK7 Plasmid Standard Curve Dilution

This is a qPCR to test the fresh p18RK7 dilution I made earlier today, and verify it works well (i.e. linear spread, good R^2 value, same Cqs as previous curve dilution, etc.).

Master mix calcs (Google Sheet): 20161128 – qPCR WS p18RK7 Curve Check

I made three separate master mixes to check the curve three times.

All samples were run in duplicate. Plate layout, cycling params, etc. are in the qPCR Report (see Results below).

Baseline threshold was manually set to 580, as previously determined by Lisa for this assay.

Results:

qPCR Report (PDF): Sam_2016-11-28 11-02-46_CC009827.pdf
qPCR Data File (CFX): Sam_2016-11-28 11-02-46_CC009827.pcrd

Overall, the curve looks good and has very comparable Cq values at each dilution of the curve to past versions of the curve. Will put new curve in the standard WS plasmid curve box in FSH240 fridge.

 

ALL MASTER MIXES

 

 

 

MASTER MIX #1


 

MASTER MIX #2

 

 


MASTER MIX #3

Share

Data Aggregation – Black Abalone qPCR Data for RLOv DNA helicase, WSN, & XC Prophage Portal Genes

Carolyn & Stand Langevin wanted some additional qPCR data for the three gene targets listed above from the 1st and 2nd black abalone experiments. I had previously aggregated dated for withering syndrome (WSN1) from the 1st black abalone experiment. Additionally, I ran qPCRs with RLOv DNA helicase and XC prophage portal genes on the black abalone samples from the 1st and 2nd experiments.

Below, is the mean Ct (Cq) and mean copy number (not applicable for XC prophage portal gene, since we don’t have a standard curve developed for this target yet) for each of the samples – sorted by abalone experiment, followed by sample accession number.

The quick summary is:

  • No phage (RLOv DNA helicase) detected in samples from 2nd black abalone experiment.
  • All but two samples (06:6-44 and 07:12-18) are positive for XC prophage portal gene.
  • Other than the 2nd black abalone experiment samples, all are positive for all three gene targets (except the two exceptions noted above).

Will email data/info to Carolyn and Stan.

I will also add this info to Lisa’s Google Sheet: Black Abalone: Expt 1 – WS & Phage. This sheet is a comprehensive collection of all the data accumulated (including histology scores, abalone gene targets, abalone morphology, etc) from the 1st abalone experiment.

 

Google Sheet: 20160425_black_ab_qPCR_gene_summaries

Share

qPCR – WSN1 & RLOv DNA helicase on Black Abalone 2nd Experiment 08:13 Accessions

Checking DNA isolated earlier today from the 2nd black abalone experiment to see if withering syndrome (RLO) and/or the withering syndrome phage (RLOv) is detectable in these samples.

Master mix calcs

Standard curves

All samples were run in duplicate.

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

Baseline thresholds were set to the following values for each assay (RLOv threshold determined by me on 20160128; WSN1 threshold determined by Lisa):

RLOv DNA helicase: 580.5

WSN1: 580

Results:

qPCR Report – RLOv DNA helicase (PDF): Sam_2016-04-21 12-39-33_CC009827_RLOv_DNA_helicase.pdf
qPCR Report – WSN1 (PDF): Sam_2016-04-21 12-39-33_CC009827_WSN.pdf
qPCR Data File (CFX): Sam_2016-04-21 12-39-33_CC009827.pcrd

RLOv DNA helicase does not amplify in any samples.

WSN1 amplifies in all samples.

All samples are RLO+/RLOv-, as seen in the previous set of 08:13 samples that I qPCR’d.

 

RLOv DNA Helicase Standard Curve

 

 

RLOv DNA Helicase Amplification (Green = Std Cuve, Blue = Samples)

 

 

 

WSN1 Standard Curve

 

WSN1 Amplification (Blue = Standard Curve, Magenta= Samples)

Share

qPCR – WSN1 & RLOv DNA helicase on Black Abalone 2nd Experiment 08:13 Accessions

Ran WSN1 and RLOv DNA helicase qPCRs on the black abalone DNA I extracted yesterday to assess whether or not these samples are RLO+/- and RLOv+/-. According to Carolyn (and this spreadsheet), they should all be RLO+/RLOv-, which is what I need in order to proceed with testing samples with the XenoCal prophage portal primers.

WSN1 Master Mix Calcs (Google Sheet): 20150330 – qPCR Black Ab 08:13 WSN1 Check

RLOv DNA Helicase Master Mix Calcs (Google Sheet): 20160330 – qPCR Black Ab 08:13 RLOv check

All samples were run in duplicate.

Plate layout, cycling params, etc. are in the qPCR Report (see Results section below).

RLOv DNA helicase standard curve from 20151224.

WSN p18RK7 standard curve from 20160316.

Baseline thresholds were set to the following values for each assay (RLOv threshold determined by me on 20160128; WSN1 threshold determined by Lisa):

RLOv DNA helicase: 580.5

WSN1: 580

Results:
qPCR Report (PDF): Sam_2016-03-30 10-00-07_CC009827.pdf
qPCR Data File (CFX96): Sam_2016-03-30 10-00-07_CC009827.pcrd

All samples are RLO+/RLOv-. This is great and can proceed with checking them with the XenoCal prophage portal primers.

 

RLOv DNA Helicase Standard Curve

 

RLOv DNA Helicase Amplification (Green = Std Cuve, Blue = Samples)

 

 

WSN1 Standard Curve

 

WSN1 Amplification (Blue = Standard Curve, Black = Samples)

Share

qPCR – Check New Withering Syndrome p18RK7 Plasmid Standard Curve Dilution

This is a qPCR to test the fresh p18RK7 dilution I made earlier today, and verify it works well (i.e. linear spread, good R^2 value, same Cqs as previous curve dilution, etc.).

Master mix calcs (Google Sheet): 20150316 – qPCR WS p18RK7 Curve Check

I made three separate master mixes to check the curve three times.

All samples were run in duplicate. Plate layout, cycling params, etc. are in the qPCR Report (see Results below).

Baseline threshold was manually set to 580, as previously determined by Lisa for this assay.

Results:

qPCR Report (PDF): Sam_2016-03-16 17-04-05_CC009827.pdf
qPCR Data File (CFX): Sam_2016-03-16 17-04-05_CC009827.pcrd

Overall, the curve looks good and has very comparable Cq values at each dilution of the curve. Will use this for future withering syndrome qPCR assays and will send an aliquote of each dilution to Colleen Burge.

MASTER MIX #1


 

MASTER MIX #2

 


MASTER MIX #3

Share

qPCR – New Withering Syndrome Phage Primers

Ran qPCR with the newly designed primers and probes for the following targets:

  • DNA Helicase (RLOv)
  • Head-to-tail gene (RLOv)
  • WSN1 (RLO)

Template DNA used:

In the histology scoring pictures below, the “New” column refers to histology scores for the presence of the phage. A score = 0 means no phage.

  • 06:5-6 (RLO only)

  • 06:6-54 (RLOv)

  • UW08:22-11A (naive pinto abalone; no RLO)

 

Master mix calcs are here: 20151008 – qPCR WS phage

All samples were run in duplicate. Cycling params, plate layout, etc. can be seen in the qPCR Report (see below).

Results:

qPCR Report (PDF): Sam_2015-10-08 17-45-38_CC009827.pdf
qPCR Data File (CFX96): Sam_2015-10-08 17-45-38_CC009827.pcrd

ORANGE – WSN1; BLUE – DNA Helicase; GREEN – Head-to-tail

The results look great! The two RLOv (phage) primer sets only amplify in the sample that has histological confirmation of the presence of phage (06:6-54). They do not amplify in the RLO-only (no phage; 06:5-6) sample, demonstrating that these two primer sets are indeed specific to the phage and don’t  amplify the RLO.

The withering syndrome primers (WSN1) were run to confirm that there aredetectable levels of RLO in both the RLOv & RLO samples, to further support the evidence showing the specificity of the two phage primer sets.

Will use the two RLOv primer sets in a conventional PCR for cloning/sequencing and development and validation of a qPCR standard curve.

Share

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.

Share