qPCR – Ava’s RLO Transmission Samples

Ran qPCRs on samples extracted earlier today. Also re-ran samples 15:08-29 and 15:09-145, per Ava’s request.

Standard curve was p18RK7 from 20161128.

All samples were run in duplicate.

Master mix calcs are here (Google Sheet): 20170619_qPCR_WSN1_Ava_Samples

Plate layouts, cycling params, etc. can be seen in the corresponding qPCR Reports (see Results below).

Baseline threshold was manually set to 580, based on Lisa’s development of the withering syndrome qPCR assay.

Results:

qPCR Report (PDF): Sam_2017-06-19 13-10-22_CC009827.pdf
qPCR Data File (CFX96): Sam_2017-06-19 13-10-22_CC009827.pcrd

 

These will need to be re-run, as the standard curve is a bit wonky (see below). Will re-run later this week.

 

DNA Extraction – Ava Withering Syndrome Transmission Study Tissues

Isolated DNA from 19 red abalone digestive gland tissue samples.

Tissue was weighed and then DNA extracted.

DNA was extracted using the QIAmp Fast DNA Stool Mini Kit (Qiagen) following the manufacturer’s protocol with the following options:

  • Samples were briefly homogenized (due to their stiffness resulting from ethanol fixation) in the InhibitEX Buffer using disposable plastic pestles.
  • Homogenized tissue was incubated at 95C to maximize cell lysis
  • Followed “human DNA analysis” protocol for remainder of protocol (to maximize sample recovery)
  • Eluted DNA with 100μL Buffer ATE

Samples were stored at 4C in FSH240 in racks for qPCR later today.

Sample information is in this spreadsheet (Google Sheet): Ava WS Transmission DNA Extractions

qPCR – Ava’s RLO Transmission Samples

Ran qPCRs on the DNA I extracted on 20170504 and earlier today.

The full list of samples is here (Google Sheet): 20170502_Ava_Ab_List

Standard curve was p18RK7 from 20161128.

All samples were run in duplicate.

Master mix calcs are here (Google Sheet): 20170509_qPCR_WSN1_Ava_Samples

Plate layouts, cycling params, etc. can be seen in the corresponding qPCR Reports (see Results below).

Baseline threshold was manually set to 580, based on Lisa’s development of the withering syndrome qPCR assay.

Results:

Curves look good on all runs (except the one that’s been noted and has been repeated). Will pass along to Ava and Carolyn.

qPCR Report (PDF): Sam_2017-05-09 07-29-36_CC009827.pdf
qPCR Data File (CFX96): Sam_2017-05-09 07-29-36_CC009827.pcrd

 


This plate has a bad curve and needs to be re-run! It has been repeated below!

I’ve included this for posterity only!

qPCR Report (PDF): Sam_2017-05-09 08-56-22_CC009827.pdf
qPCR Data File (CFX96): Sam_2017-05-09 08-56-22_CC009827.pcrd


 

 

qPCR Report (PDF): Sam_2017-05-09 10-21-15_CC009827.pdf
qPCR Data File (CFX96): Sam_2017-05-09 10-21-15_CC009827.pcrd

qPCR Report (PDF): Sam_2017-05-09 11-44-42_CC009827.pdf
qPCR Data File (CFX96): Sam_2017-05-09 11-44-42_CC009827.pcrd

 

qPCR Report (PDF): Sam_2017-05-09 13-07-46_CC009827.pdf
qPCR Data File (CFX96): Sam_2017-05-09 13-07-46_CC009827.pcrd

DNA Quantification – Ava’s RLO Transmission DNA

Quantified the DNA I isolated on 20170504 and earlier today using the Roberts Lab’s Qubit 3.0 and the dsDNA Broad Range assay.

Used 1uL of each sample.

Results:

The following samples were below the level of sensitivity of the Qubit assay:

  • 15:09-142
  • 15:11-113
  • 15:11-147
  • 15:11-149

Qubit output data (Google Sheet): 20170509_Ava_RLO_quantification_qubit

An easier-to-read summary of all the samples is here (Google Sheet): 20170502_Ava_Ab_List

 

DNA Extraction – Ava Withering Syndrome Transmission Study Tissues

Isolated DNA from five tissue samples.

Tissue was weighed and then DNA extracted.

DNA was extracted using the QIAmp Fast DNA Stool Mini Kit (Qiagen) following the manufacturer’s protocol with the following options:

  • Samples were briefly homogenized (due to their stiffness resulting from ethanol fixation) in the InhibitEX Buffer using disposable plastic pestles.
  • Homogenized tissue was incubated at 95C to maximize cell lysis
  • Followed “human DNA analysis” protocol for remainder of protocol (to maximize sample recovery)
  • Eluted DNA with 100μL Buffer ATE

Samples were stored at 4C in FSH240 in racks for qPCR and quantification later today.

Sample information is in this spreadsheet (Google Sheet): 20170502_Ava_Ab_List

DNA Extraction – Ava Withering Syndrome Transmission Study Tissues

Isolated DNA from 117 tissue samples that I weighed out yesterday.

DNA was extracted using the QIAmp Fast DNA Stool Mini Kit (Qiagen) following the manufacturer’s protocol with the following options:

  • Samples were briefly homogenized (due to their stiffness resulting from ethanol fixation) in the InhibitEX Buffer using disposable plastic pestles.
  • Homogenized tissue was incubated at 95C to maximize cell lysis
  • Followed “human DNA analysis” protocol for remainder of protocol (to maximize sample recovery)
  • Eluted DNA with 100μL Buffer ATE

Samples were stored at 4C in FSH240 in racks for qPCR and quantification next week.

Sample information is in this spreadsheet (Google Sheet): 20170502_Ava_Ab_List

DNA Quantification – RLO viability DNased RNA

I previously DNased RNA I isolated from water filters that were part of the RLO viability experiment that Lisa and the Capstone students are conducting. I checked for residual gDNA carryover via qPCR and all of the samples that were intended for dosing the abalone came up positive. It’s likely due to such a high quantity of algae that was co-filtered with the potential RLOs, leading to over-saturation of the RNAzol with DNA, resulting in the gDNA carryover.

In turn, I think the DNase treatment was insufficient for the quantity of carryover DNA.

I am planning on re-DNasing those samples, but want to quantify any residual DNA present to make sure that the samples aren’t still too concentrated for the DNase.

Samples were quantified using the Robert Lab Qubit 3.0 and the Qubit dsHS reagents (high sensitivity), using 1uL of sample.

Results:

Residual DNA is still present, but at levels that are well below the maximum that the DNase treatment (10ug) can handle. I will redo the DNase treatment on these samples. Spreadsheet is linked, and embedded below, with sample concentrations.

Spreadsheet (Google Sheet): 20170424_filter_rna_dna_quant

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

 

 

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)