Data Summary – Black Abalone Phage qPCRs

A quick summary table of the various black abalone qPCRs I ran yesterday:

SAMPLE RLO_MCP RLO_ph_protease XC_prophage_portal RLOv_DNA_helicase WSN
06:06-50  +  +  +  +  +
06:06-52  +  +  +  +  +
07:12-01  -  -  -  +  -
07:12-02  -  -  -  -  -
08:13-05  +  +  +  -  +
08:13-18  +  +  +  -  +
08:13-24  +  +  +  -  +*
08:13-25  +  +  +  -  +
  • This sample technically showed amplification, but came up after the last point on the standard curve. Most likely due to extremely low concentration (~0.5ng/uL).

  • RLO Major Capsid Protein (RLO_MCP)

  • RLO Prohead Protease Protein (RLO_ph_protease)
  • XenoCal Phage Portal Gene (XC prophage)

qPCR – WSN on Black Abalone

Ran qPCRs on a set of black abalone digestive gland DNA (sample list provided by Carolyn):

07:12-01 (Black Ab exp 1)
07:12-02 (Black Ab exp 1)
08:13-05 (Black Ab exp 2)
08:13-18 (Black Ab exp 2)
08:13-24 (Black Ab exp 2)
08:13-25 (Black Ab exp 2)
UW06:06-32
UW06:06-41
UW06:06-50 (Black Ab exp 1)
UW06:06-52 (Black Ab exp 1)

The two samples with a strikethrough did not have any DNA left in the tubes and were not run.

All samples were run in duplicate.

Standard curve was p18RK7 from 20161128.

Cycling params, plate layout, etc can be seen in the qPCR Report (see Results).

Baseline was set 580 as previously determined by Lisa.

Results:
qPCR Report (PDF): Sam_2017-04-13%2016-20-54_CC009827_WSN1.pdf
qPCR Data File (CFX): Sam_2017-04-13%2016-20-54_CC009827_WSN1.pcrd

Standard curve looked good.

The following samples did not amplify:
– 07:12 set
– Note: 08:13-24 technically did amplify, but comes up below the lowest point of the standard curve, so technically it is effectively “no amplification”.

The remaining samples all came up positive.

Will convey to Carolyn and Stan.

qPCR – RLOv DNA Helicase on Black Abalone

Ran qPCRs on a set of black abalone digestive gland DNA (sample list provided by Carolyn):

07:12-01 (Black Ab exp 1)
07:12-02 (Black Ab exp 1)
08:13-05 (Black Ab exp 2)
08:13-18 (Black Ab exp 2)
08:13-24 (Black Ab exp 2)
08:13-25 (Black Ab exp 2)
UW06:06-32
UW06:06-41

UW06:06-50 (Black Ab exp 1)
UW06:06-52 (Black Ab exp 1)

The two samples with a strikethrough did not have any DNA left in the tubes and were not run.

All samples were run in duplicate.

Standard curve was from 20161106.

Cycling params, plate layout, etc can be seen in the qPCR Report (see Results).

Baseline was set 580.5 as previously determined.

Results:
qPCR Report (PDF): Sam_2017-04-13%2016-20-54_CC009827_RLOv_helicase.pdf
qPCR Data File (CFX): Sam_2017-04-13%2016-20-54_CC009827_RLOv_helicase.pcrd

Standard curve looked good, although efficiency is pushing it on the high end.

The following samples did <em>not</em> amplify:

  • 07:12-02
  • All 08 samples.

The remaining samples all came up positive, with the 06 set being extremely hot (came up around cycle 13).

Will convey to Carolyn and Stan.

 

 

qPCR – RLO Prophage Genes

Ran qPCRs on a set of black abalone digestive gland DNA (sample list provided by Carolyn):

07:12-01 (Black Ab exp 1)
07:12-02 (Black Ab exp 1)
08:13-05 (Black Ab exp 2)
08:13-18 (Black Ab exp 2)
08:13-24 (Black Ab exp 2)
08:13-25 (Black Ab exp 2)
UW06:06-32
UW06:06-41

UW06:06-50 (Black Ab exp 1)
UW06:06-52 (Black Ab exp 1)

The two samples with a strikethrough did not have any DNA left in the tubes and were not run.

Gene targets:
– RLO Major Capsid Protein (RLO_MCP)
– RLO Prohead Protease Protein (RLO_ph_protease)
– XenoCal Phage Portal Gene (XC prophage)

Master mix calcs are here (Google Sheet): 20170413 – qPCR_XCphagePortal_RLOcapsid_RLOprohead

The same master mix calculations were used for each, just swapped in appropriate primers.

All samples were run in duplicate.

Cycling params, plate layout, etc. can be found in the qPCR Report (see Results below).

Results:
qPCR Report (PDF): Sam_2017-04-13 14-56-03_CC009827.pdf
qPCR Data File (CFX): Sam_2017-04-13 14-56-03_CC009827.pcrd

Melt curves for all three primer sets looked perfect (see below)

Amplification present for all samples, with all three primers except the 07:12 samples.

Will pass info along to Carolyn and Stan.

Will add info to the following two spreadsheets (Google Sheets):

Black Abalone: Expt 1 – WS & Phage

Black Abalone: Expt 2 – WS only

 


 

Green = RLO_ph_protease

Brown = RLO_MCP

Blue = XC_prophage

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

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

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.

 

 

 

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.

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)

DNA Isolation – Black Abalone 2nd Experiment 08:13 Accessions

Isolated DNA from EtOH-preserved black abalone digestive gland tissue from the 2nd black abalone experiment.

There’s some odd background in regards to these samples which I previously described here that might be worth reviewing.

DNA was isolated using the QIAamp Fast DNA Stool Kit (Qiagen). Tissues were weighed and briefly homogenized with a disposable pestle in InhibitEX Buffer. Manufacturer’s protocol was followed. DNA  was eluted in 100μL of Buffer ATE and quantified on the Roberts Lab Qubit3.0 (ThermoFisher) using 1μL with the Qubit dsDNA Broad Range assay.

Results:

Google Sheet: 20160421_DNA_isolation_08:13_subset