Tag Archives: RLOv_DNA_helicase

qPCR – RLOv DNA helicase and XenoCal prophage on Ab Endo Water Filters

Stan Langevin was interested in seeing if the RLOv (phage) and/or the prophage portal genes were detectable in water samples from Lisa’s Ab Endo project.

Ran qPCR on the following samples that Lisa selected:

DNA from water filters collected in 2010. DNA isolated 20120111:

  • CP 0M A
  • CP 0M B
  • MA 0M A
  • MA 0M B
  • PSN 0M A
  • PSN 0M B
  • RM A
  • RM B

DNA from water filters collected in 2011. DNA isolated 20140822:

  • AM Drain 2B
  • PCI SRI PC 1B

RLOv_DNA_helicase master mix calcs are here (Google Sheet): 20161213 – qPCR RLOv DNA Helicase

XenoCal prophage master mix calcs are here (Google Sheet): 20161213 – qPCR XenoCal phage portal

RLOv_DNA_helicase standard curve from 20151224.

All samples were run in duplicate. Plate layout, cycling params, etc. can be seen in the qPCR Report below.

Results:

RLOv_DNA_helicase
qPCR Report (PDF): Sam_2016-12-13 14-52-05_CC009827_RLOv_helicase.pdf
qPCR Data File (CFX): Sam_2016-12-13 14-52-05_CC009827_RLOv_helicase.pcrd

 

XenoCal prophage
qPCR Report (PDF): Sam_2016-12-13 14-52-05_CC009827_XCprophage.pdf
qPCR Data File (CFX): Sam_2016-12-13 14-52-05_CC009827_XCprophage.pcrd

 

  • RLOv DNA helicase amplified in all samples EXCEPT the two samples from 2011. These two samples were negative for the RLO (see Ab Endo sheet “water 2011″).
  •  XC prophage amplfied inconsistently (i.e. replicates did not match/amplify) in only three samples. Additionally, the melt curve of one of those samples differs from the other two. Based on the inconsistencies in technical reps, I should probably repeat this, but technical reps across all of the RLOv DNA helicase samples are very tight, suggesting that my technique was fine (it would be odd if my technique faltered only on ALL of the XC prophage samples)…

 

RLOv DNA HELICASE

 


 

XENOCAL PROPHAGE

 

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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

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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

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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)

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Data Aggregation – WS RLO and RLOv DNA Helicase qPCR and WS RLO Infection Intensities

Carolyn asked me to send her the data described above.

RLOv DNA helicase qPCR data were grabbed from the qPCR I ran on 20160106.

The qPCR data for withering syndrome RLO were culled from these three different spreadsheets:

 

The summary is below. I have emailed a copy of the spreadsheet to Carolyn.

Google Sheet: 20160404_Summary_RLO_RLOvDNAhelicase_qPCR_HistoIntensities

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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)

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qPCR – Repeat Phage Portal Primer Specificity Check

This should be the last time I run this for the time being. Re-running this with undiluted RLOv- samples to improve the melt curve resolution for better comparison to the RLOv+ melt curves.

See the earlier qPCR run for master mix calcs.

All samples were run in duplicate.

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

Results:

qPCR Report (PDF): Sam_2016-03-17 13-30-29_CC009827.pdf
qPCR Data File (CFX): Sam_2016-03-17 13-30-29_CC009827.pcrd

These results are interesting and I believe they are real, as opposed to the confusing/conflicting information I got from the previous two qPCRs from earlier today with the phage portal gene primers.

The poor/confusing results from the two previous qPCR attempts seem to have stemmed from low sample concentration. Using undiluted RLOv- samples in this run has resulted in clear, definitive data.

The melt curves are of a single peak in all RLOv+ samples.

The phage portal gene is NOT detected in RLOv- samples. However, it is present in RLOv+ samples and at significantly lower abundance than the RLOv DNA helicase (DNA helicase comes up at ~23 Cqs in samples that have been diluted 1:1000, while the phage portal gene comes up at ~28 Cqs in UNDILUTED samples). Alternatively, it is possible that the phage portal qPCR is less efficient and/or is experiencing some sort of inhibition; both seem unlikely, though.

Will discuss with Carolyn to see if she wants to go forward with cloning/sequencing and construction of a plasmid standard curve for the phage portal gene.


qPCR Amplification Plots (DNA helicase in green; Phage portal gene in blue)

 


 

qPCR Amplification Plots of Phage Portal Gene

 

qPCR Melt Curves of Phage Portal Gene

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qPCR – Repeat Phage Portal Primer Specificity Check

Due to the odd (and poor) results from the first qPCR to look at this phage portal gene, I’m repeating the qPCR exactly, but have made fresh 1:1000 dilutions of the two RLOv+ samples (08:3-15, 08:3-16) with TE.

See the earlier qPCR run for master mix calcs.

All samples were run in duplicate.

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

Results:

qPCR Report (PDF): Sam_2016-03-17 10-42-54_CC009827.pdf
qPCR Data File (CFX): Sam_2016-03-17 10-42-54_CC009827.pcrd

Firstly, the fresh dilutions resolved the issue with the poor amplification previously seen with the RLOv DNA helicase assay; they look perfect in this run.

Quick summary for the phage portal qPCR:

  • Amplification in all 4 samples (RLOv- & RLOv+).
  • Much, much earlier amplification in RLOv+ samples.
  • Good, single peaks in melt curves
  • RLOv+ and RLOv- samples show different temps for peaks in melt curves

If the phage portal gene was present in the RLOv, then we would expect the amplification (i.e. the Cq values) of DNA helicase and the phage portal gene to be extremely close. However, in the RLOv+ samples, there’s a ~1000-fold difference in DNA helicase/phage portal levels.

If the phage portal gene was present in just the RLO, then we would expect similar amplification (i.e. Cq values) in both RLOv+/- samples. However, we see an EXTREME difference in phage portal gene levels between RLOv+ samples and the RLO- samples (~10,000-fold difference in levels). If the phage portal gene was present in both RLOv+/- samples, then that could possibly help explain this difference, due to the massive phage load in the RLOv+ samples (based on DNA helicase data). However, this doesn’t seem to be the case…

We see two distinct melt curve peak temps between the RLOv+/- samples. If the phage portal gene was present in both sample types, then the RLOv+ samples should exhibit a dual peak in the melt curves. However, this is not the case. This is difficult to explain since the RLOv+ samples also contain RLOv- (i.e. the RLO bacteria) DNA. If the PCR product generated in the RLOv- samples is indeed distinct from that produced in the RLOv+ samples, then we should see that product in the melt curve of the RLOv+ samples, but we don’t.

I will repeat this qPCR using undiluted, source DNA from the RLOv- samples. This should shift their amplification ~10 Cq (a 10-fold difference in amplification equates to ~3.32 Cqs) earlier. This, in turn, will allow their signal to generate higher levels of fluorescence and, hopefully, increase the melt curve peak for a more accurate assessment of melt temp(s); just to make sure the melt temp is accurate.


 

qPCR Amplification Plots (DNA helicase in green; Phage portal gene in blue)


qPCR Amplification Plots of Phage Portal Gene

qPCR Melt Curves of Phage Portal Gene

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qPCR – Phage Portal Primer Specificity Check

Stan Langevin recently identified a RLOv phage portal protein sequence that he wanted to see if this gene is incorporated in the withering syndrome bacteria (RLO) or the phage (RLOv). He designed a primer and probe set:

I’ve ordered/received the primers and need to test them out verify their specificity (via melt curve analysis) to ensure they only amplify a single target before proceeding with the expense of ordering a probe, as well as the time/effort that will be needed to, potentially, create a plasmid standard curve.

Set up qPCR on the following samples using the new RLOv phage portal primer set, as well as the RLOv DNA helicase qPCR assay to serve a positive control. These samples have been previously qPCR’d with the RLOv DNA helicase assay to establish the presence/quantities of RLOv in these samples. Samples are 1:1000 dilutions of the source DNA (made 20160106, but made fresh dilutions of RLOv negative samples today in H2O) due to the extremely high levels of RLOv detected in the RLOv positive samples.

SAMPLE RLOv +/-
08:4-3 NEGATIVE
08:4-4 NEGATIVE
08:4-15 POSITIVE
08:4-16 POSITIVE

 

RLOv phage portal master mix calcs (Google Sheet): 20160317 – qPCR XenoCal phage portal specificity

RLOv DNA Helicase master mix calcs (Google Sheet): 20160317 – qPCR RLOv

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

Results:

qPCR Report (PDF): Sam_2016-03-17 08-03-13_CC009827.pdf
qPCR Data File (CFX): Sam_2016-03-17 08-03-13_CC009827.pcrd

The results are very odd and the qPCR should be repeated with fresh dilutions of the source DNA. Here are the reasons:

  • Almost no amplification with DNA helicase in RLOv+ samples
  • Amplification in all but one sample with phage portal – expected in all, none, RLOv- only, or RLOv+ only
  • Phage portal melt curves differ across samples – even within same sample type (i.e. RLOv-
  • Multiple peaks in phage portal melt curve in one RLOv- sample
  • Single peak in phage portal melt curve in the RLOv+ sample

See the amplification plots and melt curves below for a better idea of what’s happening.

 


qPCR Amplification Plots (DNA helicase in green; Phage portal gene in magenta)


 

 

qPCR Amplification Plots of Phage Portal Gene

 

qPCR Melt Curves of Phage Portal Gene

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Data Analysis – RLOv DNA Helicase qPCR Limit of Detection Calcs

I previously ran three qPCR plates (20160121, 20160122, & 20160125) with 20 reps of each of the following copy numbers to determine the limit of detection (or, analytical sensitivity) of this qPCR assay: 30, 10, 3, 1

Additionally, I determined the average baseline threshold to set (580.5), based on eight qPCRs (see 20160128).

Performed calculations and determined limit of detection (defined as >95% of samples produced amplification) for this assay is three copies. The single copy sample amplified 88% of the time. This also means that our cycle threshold cut-off for this qPCR assay should be 37.9, as the mean Cq for three copies was 37.33, with a standard deviation of 0.53.

The data and calculations can be seen below (Google Sheet – scroll to the right to see calcs):

Google Sheet: RLOv_DNA_helicase_LoD_calcs

 

 

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