Tag Archives: qPCR

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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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…

 

 

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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.

 

 

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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

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Data Management – Script for Compiling qPCR Data

Over the last few weeks, I’ve wrestled with tracking down data (primarily qPCR data) from the litany of projects the Friedman Lab has had over the last decade or so. I’ve also noticed that it’s increasingly difficult for me to track down my own data from individual projects where data is not generated continuously over time, but in chunks. Tracking down 6 different qPCR runs that were conducted over the course of a year is tedious.

In order to save myself, and others who might need/want to review my data, a lot of time in the future, I decided to write a script that will allow me to compile all of my qPCR data into a single, massive CSV file. Accessing this CSV file via spreadsheet (Excel/Calc/Sheets) or database (SQL) means I’ll always be able to quickly search for all the related data I need, since it will reside in a single file!

The script is written in bash, called qpcr_aggregation.sh, and is currently hosted here: https://github.com/kubu4/Scripts/blob/master/bash/qpcr_aggregation.sh

Basically, the script does the following:

  • Replace the spaces in the BioRad filenames with underscores (used to simplify parsing of the filename for use in downstream steps in the script)
  • Replace the header row to accommodate two new fields: qPCR_filename and qPCR_date
  • Add qPCR_filename and qPCR_date to each file.
  • Concatenate all the files into a single “master” CSV file.

Now, the easy part – exporting the data from hundreds of qPCR files…

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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 – Black Abalone with XC Prophage Portal Primers

I accidentally skipped two samples from the 2nd black abalone experiment sample set that I qPCR’d last week, so I’m qPCRing them today.

Master mix calcs (Google Sheet): 20160425 – qPCR Black Abs XenoCal phage portal

All samples were run in duplicate.

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

Results:
qPCR Report (PDF): Sam_2016-04-25 12-55-40_CC009827.pdf
qPCR Data File (CFX96): Sam_2016-04-25 12-55-40_CC009827.pcrd

Have amplification in both samples.

I will add this to a “master” spreadsheet that I’ve made containing qPCR data from three genes on ~20 samples from both the 1st and 2nd black abalone experiments.

 

 

 

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qPCR – Black Abalone with XC Prophage Portal Primers

Ran qPCR with black abalone samples from the 1st and 2nd experiments to see if the Xenocal prophage portal gene is detected.

Master mix calcs (Google Sheet): 20160421 – qPCR Black Abs XenoCal phage portal

All samples were run in duplicate.

Black abalone sample 08:13-2 was run as a positive control.

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

Results:
qPCR Report (PDF): Sam_2016-04-21 14-11-09_CC009827.pdf
qPCR Data File (CFX): Sam_2016-04-21 14-11-09_CC009827.pcrd

Two samples failed to produce amplification: 06:6-44 and 07:12-18. All other samples amplified. Will compile this data with WSN and RLOv DNA helicase and send along to Carolyn and Stan.

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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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