Tag Archives: Dg

qPCR – CDFW White Abalone Samples (RLOv DNA helicase)

The samples that CDFW sent us earlier were previously checked for RLO presence with the withering syndrome qPCR assay.

Standard curve was from 20151106.

All samples were run in duplicate.

Master mix calcs are here; since I ran these with the other samples, the master mix used was part of the other project indicated in the spreadsheet (Google Sheet): 20170420 – qPCR RLOv DNA Helicase

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

Baseline threshold was manually set to 580.5, as previously determined.

Results:

qPCR Report (PDF): Sam_2017-04-20 07-50-18_CC009827.pdf
qPCR Data File (CFX): Sam_2017-04-20 07-50-18_CC009827.pcrd

Standard curve looks good and all samples provided come up positive for RLOv DNA helicase.

I’ve compiled the raw data of both the WSN qPCR and this in this Google Sheet: 20170420_CDFW_White_Ab_qPCR_summary

Here’s a summary table of the results (copy numbers are mean copies from qPCR replicates):

SAMPLE RLOV DNA HELICASE (COPIES) WSN1 (COPIES)
SF16-76_DG-1  165318.58 169.25
 SF16-76_DG-2  47839.81  20.70
 SF16-76_PE-1  1036697.17 633.75
 SF16-76_PE-2  46763.60  296.83
 SF17-17  117.29  2.16

NOTE: The WSN1 copies for SF17-17 is below the accepted detection limit of the qPCR assay (i.e. < 3 copies).

Will share my notebooks and spreadsheet with Blythe at CDFW.

Amplification Plots

Green = Standard Curve

Blue = Samples

Red = No template control

 

 

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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

 

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

Oddly, I was unable to find any DNA for the 08:13 samples that should have been previously qPCR’d for RLO.

Instead, I tracked down the EtOH-preserved digestive gland (DG) tissues from when these were initially sampled. The box contained both of the “QPCR” tissue samples, however, many of them had dried out. This fact had already been denoted on the outside of the box and on the tubes.

Finding these samples is a bit strange. It’s odd because if someone had performed qPCR analysis on these 08:13 samples, the DNA should’ve come from either of the two “QPCR” tissue samples; but, looking at the vials, it seems like no tissue has been removed from any of the tubes…

Additionally, despite the fact that the spreadsheet Carolyn provided me with the other day indicating that the 08:13 samples are from the 2nd black abalone experiment, the label on this box indicates that these are from the 1st black abalone experiment… Despite this, I’m fairly certain these are indeed from Experiment 2, as these accession numbers have never been brought up before in any of Lisa’s extensive work on the 1st black abalone experiment.

I extracted DNA using the QIAmp Fast DNA Stool Mini Kit (Qiagen) from the following samples. DNA was eluted with 100μL of Buffer ATE and quantified on the Roberts Lab Qubit3.0 (ThermoFisher) using 1μL.

ACCESSION
08:13-2
08:13-3
08:13-4
08:13-5
08:13-6
08:13-7
08:13-11
08:13-12
08:13-13
08:13-14
08:13-16
08:13-17

Results:

Google Sheet: 20160329_DNA_isolation_08:13_subset

Will run qPCRs (WSN1, RLOv DNA helicase, and XenoCal prophage portal) on these samples tomorrow.

DNA has been stored in an existing box in the full-sized -20C freezer in FSH240 and the label on the box has been updated to include these samples.

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Sample ID – Black Abalone DNA for RLOv qPCRs

Carolyn & Stan Langevin have agreed that the DNA helicase qPCR should be tested on 10 black abalone DNA extractions that fall into multiple levels of the Friedman Lab’s withering syndrome histology scoring.

Downloaded the (Google Sheet) Black Abalone: Expt 1 – WS & Phage as a CSV file. After downloading, I renamed the file (Black_Abalone.csv) to facilitate easier usage in the following steps.

Created a sqlite database using GitBash for Windows:
Change to directory where file is located:

$cd Downloads

Start sqlite:

$sqlite3

Tell sqlite that the field separator will be commas (i.e. CSV file):

sqlite>.separator ","

Import the CSV file and provide a name for the resulting database:

sqlite>.import Black_Abalone.csv BlackAbs

Set output display mode to column for easier reading:

sqlite>.mode column

Set output display to include column headers:

sqlite>.headers on

 

To select all the samples that have scores of 0 in both PE and DG RLO fields (screen cap does not show entire output list):

 

To select all the samples that have scores of 1 in both PE and DG RLO fields:

 

To select all the samples that have scores of 2 in both PE and DG RLO fields:

 

Here are the full set of results in a table

RLO/RLOv 0 RLO/RLOv 1 RLO/RLOv 2
06:5-03 06:5-35A 06:5-31
06:5-04 06:50-08 06:5-32B
06:5-08 06:50-10 06:6-46
06:5-09 06:6-32 06:6-49
06:5-10 06:6-39 08:3-05
06:5-11 06:6-42 08:3-07
06:5-14 06:6-44 08:3-15
06:5-16 06:6-52 08:3-16
06:5-18 06:6-54
06:5-20 07:12-18
06:5-21 08:3-08
06:5-22 08:3-10
06:5-24
06:5-30
06:50-04
06:50-05
06:50-11
06:50-12
06:50-13
06:50-15
06:50-16
06:6-01
06:6-02
06:6-03
06:6-05
06:6-08
06:6-11
06:6-12
06:6-13
06:6-15
06:6-16
06:6-17
06:6-18
06:6-20
06:6-21
06:6-22
06:6-23
06:6-24
06:6-25
06:6-26
06:6-27
06:6-28
07:12-01
07:12-02
07:12-03
07:12-04
07:12-05
07:12-06
07:12-07
07:12-09
07:12-10
07:12-13
07:12-19
08:3-01
08:3-02
08:3-03
08:3-04
08:3-13
08:4-01
08:4-02
08:4-03
08:4-04
08:4-05
08:4-06
08:4-07
08:4-08
08:4-09
08:4-10
08:4-11
08:4-12
08:4-13
08:4-14
08:4-15
08:4-16
08:4-17
08:4-18
08:4-19
08:4-20
08:4-21
08:4-22
08:4-23
08:4-24
08:4-25
08:5-06

Will select just 10 of those in the RLO/RLOv 0 column for use in qPCR.

I was able to track down the boxes where are these DNAs were stored (see images below).

Boxes that were not labeled with accession numbers of the samples contained therein are now labeled.

Boxes that contained samples that belonged in other boxers were transferred to the appropriate box.

All boxes were located, and returned, to the big -20C in 240 on Lisa’s shelf.

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Abalone Sampling – Post-esophagus & Digestive Gland

Helped Ava sample 162 red abalone (Haliotis rufescens).

Accession numbers 15:11-1 through 15:11-162

  • Animals were weighed.
  • Took one post-esophagus sample and one digestive gland sample for histology, placed in histology cassette; three animals per cassette.
  • Took one post-esophagus sample in 95% ethanol for qPCR
  • Shells were labeled and retained for post-sampling weighing/measurements.

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Abalone Sampling – Post-esophagus & Digestive Gland

Helped Ava sample 74 red abalone (Haliotis rufescens).

Accession numbers 15:10-1 through 15:10-74

  • Animals were weighed.

  • Took one post-esophagus sample and one digestive gland sample for histology, placed in histology cassette; three animals per cassette.

  • Took one post-esophagus sample in 95% ethanol for qPCR

  • Shells were labeled and retained for post-sampling weighing/measurements.

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Abalone Sampling – Post-esophageal & Digestive Gland Tissues

Ava sent up abalone for sampling from California for tissue sampling. Here’s the summary of how things went.

  • Animal temps @ -8.0C when opened at lab
  •  ~120 animals sampled
  •  Only 1 dead animal found- Additional dead abalone included in shipment were NOT sampled; too dead
  • Started @ ~11AM, finished at 5:15PM (3 people dissecting, 1 person weighing)
  • All mesh bags were saved and are in a 10% bleach solution over night in sink in 236
  • Histo cassettes put in Davidson’s over night; need to be transferred to 70% EtOH tomorrow
  • Histo cassette layout: animal #1 upper left, animal #2 upper right, animal #3 lower left
  • Tubes for 15:9 sampling have labels applied
  • Histo cassettes for 15:9 sampling have NOT been labelled
  • Empty shells have been labelled and saved; will need to be weighed/measured later

 

 

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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.

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qPCR – Evaluation of withering syndrome and phage presence in holding tanks

In anticipation of receiving a large quantity of abalone from Japan, Carolyn wants to assess  current infection status of our abalone to make decisions on how/where to house the incoming abalone.

Ran a qPCR to detect withering syndrome and the withering syndrome phage on the following DNA samples isolated today by Lisa:

  • RR1 (Haliotis discus discus) – Seawater DNA
  • RR2 (Haliotis diversicolor) – Seawater DNA
  • 14:5-1 – 4- Dg DNA

Positive control: pCR2.1/ORF25 (1:1000) from 20141008.

Primers used:

  • Withering Syndrome – WSN1F/R
  • Phage – 1_ORF25F_225_CSF, 1_ORF25R_399_CSF,

All samples were run in duplicate.

Master mix calcs are here: 20150316 – qPCR H.discus H2O and feces

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

Results:
qPCR Report (PDF): Sam_2015-03-16 16-46-06_CC009827.pdf
qPCR Data File (CFX96): Sam_2015-03-16 16-46-06_CC009827.pcrd

Withering syndrome
– standard curve is perfect
– all samples, except seawater RR2, amplified

Phage
– no standard curve; this is not ready yet; as such, you can ignore the copy number (SQ) listed in the data file
– consistent amplification in both seawater samples (RR1, RR2)
– zero or inconsistent (i.e. one of two reps amplified) in remaining samples
– melt curves in the RR1 and RR2 samples exhibit multiple peaks, suggesting amplification of multiple targets (i.e. lack specificity)
– melt curves in the remaining samples only exhibit single peaks
– RR2 melt curves are shifted 2C (82C), compared to all other samples (80C)

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