Tag Archives: RLO

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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Reverse Transcription – Water Filter DNased RNA (from 20161207)

Performed reverse transcription on the DNased RNA samples that I verified were free of detectable RLO DNA (20161207).

Combined 17μL DNased RNA + 0.5μL random primers (Promega; Cat: C1181) in 0.2mL PCR tubes.

NOTE: The 17μL was virtually all of the sample volume recovered from DNasing. As such, the DNased RNA will not be quantified.

Incubated DNased RNA and primer mix in PTC-200 thermal cycler (MJ Research) at 70C for 5mins w/heated lid, then immediately placed on ice.

Created master mix of following components:

REAGENT SINGLE REACTION VOL (μL) NUMBER REACTIONS TOTAL VOL (μL)
5x MMLV RT BUFFER 5 14 70
10mM dNTPS (Promega) 1.25 14 17.5
MMLV RT (Promega) 0.5 14 7

Added 6.75 of master mix to each and mixed by pipetting.

Incubated PTC-200 thermal cycler (MJ Research) @ 37C for 1hr (no heated lid), followed by 95C for 3mins (heated lid). Samples were transferred to 0.5mL snap cap tubes and labelled with “cDNA” and the corresponding sample name. Samples will be stored in my -20C box.

UPDATE 20170830 Lisa has moved these samples to a -20C box dedicated to RLO Viability cDNA.

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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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DNase Treatment – Water Filter RNA from 20161130

Continued preparation of this RNA to assess withering syndrome viability in the water column. I treated the RNA I isolated on 20161130 using the Turbo DNA-free (Ambion) DNase kit, according to their protocol.

Added the following to each sample:

  • 2.5μL 10x buffer
  • 1.5μL H2O
  • 1μL DNase

Incubated @ 37C for 1hr.

Added 0.1 volumes (2.5μL) of DNase Inactivation reagent and incubated at RT for 2mins (with mixing). Pelleted inactivation reagent: 10,000g, 2mins, RT. Transferred supe to new tube.

Samples were labelled as “DNased RNA”, their existing sample name (see below), and stored @ -80C.

Sample names:

  • T0A
  • T0B
  • T1A
  • T1B
  • T1A C
  • T1B C
  • T3A
  • T3B
  • T7A
  • T7B
  • T7A C
  • T7B C
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RNA Isolation – Abalone Water Filters

Lisa provided me with a set of water filters stored in 1mL RNAzol RT @ -80C to isolate RNA from. This is an attempt to assess withering syndrome viability from within the water.

The samples were thawed and briefly homogenized (as best I could) with a disposable plastic pestle. The samples were then processed according to the manufacturer’s protocol for total RNA isolation. Samples were resuspended in 20μL of 0.1%-DEPC H2O.

Samples were stored @ -80C. Will DNase next week.

The sample names are as follows (the ‘C’ is short for “Control”, per Lisa):

  • T0A
  • T0B
  • T1A
  • T1A C
  • T1B C
  • T3A
  • T3B
  • T7A
  • T7B
  • T7A C
  • T7B C

 

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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 – XenoCal Prophage Portal Primers on RLO/RLOv positive/negative samples

Stan Langevin and Carolyn wanted to see if this particular gene was found in the withering syndrome (RLO) or phage (RLOv) genomes. I previously identified 10 samples of each of the following combinations:

  • RLO-/RLOv-
  • RLO-/RLOv+
  • RLO+/RLOv-
  • RLO+/RLOv+

Master mix calcs are here (Google Sheet): 20160331 – qPCR Black Ab 08:13 XenoCal phage portal check

All samples were run in duplicate.

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

Results:
qPCR Report (PDF): Sam_2016-03-31 08-51-11_CC009827.pdf
qPCR Data File (CFX): Sam_2016-03-31 08-51-11_CC009827.pcrd

The results are definitely interesting!

Quick summary: Amplification only seen in RLO+/RLOv- samples!

Oddly, there is no amplification in the other group of RLO+ samples (RLO+/RLOv+). Based on the fact that there is amplification in RLO+/RLOv- samples, which implies this prophage portal gene is present in withering syndrome, we would expect to also have amplification in the other group of samples that are positive for withering syndrome.

Compiled qPCR data with WSN1, RLOv_DNA_helicase, and XenoCal prophage portal gene (Google Sheet): 20160331_qPCR_summary_RLO_RLOv_pos_negs

 

Amplification Plots (PINK= RLO+/RLOv-, BLUE = RLO-/RLOv-; GREEN = RLO-/RLOv+; BLACK = RLO+/RLOv+)

 

Melt Curve Plots (see amplification plots for color scheme)

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Sample ID- XenoCal Prophage Portal Tests

Now that the XenoCal prophage portal primers appear to be in working order, Carolyn wants me to test them out on 10 samples with the following status':

  • RLO-/RLOv-
  • RLO-/RLOv+
  • RLO+/RLOv-
  • RLO+/RLOv+

In order to quickly identify samples with these qualifications, I ran a SQL query on the following spreadsheet that contain qPCR data for both withering syndrome (RLO) and the phage (RLOv):

I saved the following worksheets from the above Google Sheet as CSV files:

  • water 2010
  • water 2011

These were imported to SQLite as I’ve previously done.

The two sheets were renamed for use in SQLite, respectively:

  • AbEndoWater2010
  • AbEndoWater2011

Here are the four queries I ran to obtain the four combinations of RLO/RLOv samples listed above

RLO-/RLOv-

sqlite> SELECT '2011_H2O', "DNA Tube Label", "Mean Cq", "RLOv_mean_Cq" FROM AbEndoWater2011 WHERE "Mean Cq"=0 AND "RLOv_mean_Cq"=0

 

RLO-/RLOv+

sqlite> SELECT '2011_H2O', "DNA Tube Label", "Mean Cq", "RLOv_mean_Cq" FROM AbEndoWater2011 WHERE "Mean Cq"=0 AND "RLOv_mean_Cq">0

 

RLO+/RLOv-

sqlite> SELECT '2011_H2O', "DNA Tube Label", "Mean Cq", "RLOv_mean_Cq" FROM AbEndoWater2011 WHERE "Mean Cq">0 AND "RLOv_mean_Cq"=0

 

RLO+/RLOv+

sqlite> SELECT '2011_H2O', "DNA Tube Label", "Mean Cq", "RLOv_mean_Cq" FROM AbEndoWater2011 WHERE "Mean Cq">0 AND "RLOv_mean_Cq">0

 

Results:

It looks like we do not currently have 10 samples that are RLO+/RLOv-. I will contact Carolyn to see if she happens to know of any samples that are RLO+, but do not contain (or, should not) any RLOv.

The full list of results can be seen in the Google Sheet below.

Google Sheet: 20160322_RLO_RLOv_pos_negs

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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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