qPCR – Pinto Abalone DNA with WSN1 and RLOv DNA Helicase

Ran qPCRs using both WSN1 and RLOv DNA Helicase primers on the pinto abalone DNA isolated earlier today, as well as one additional sample that Sean Bennett had previously isolated DNA from: 15:6-26E

RLOv helicase standard curve is from 20151106.

WSN1 standard curve is p18RK7 from 20170703

All samples were run in duplicate.

Master mix calcs (Google Sheet): 20171226 – qPCR Pinto WSN1 & RLOv DNA Helicase.

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

Results:

RLOv helicase qPCR Report (PDF): Sam_2017-12-26 13-19-51_CC009827_RLOv_helicase.pdf
RLOv helicase qPCR File (CFX): Sam_2017-12-26 13-19-51_CC009827_RLOv_helicase.pcrd

WSN1 qPCR Report (PDF): Sam_2017-12-26 13-19-51_CC009827_WSN1.pdf
WSN1 qPCR File (CFX): Sam_2017-12-26 13-19-51_CC009827_WSN1.pcrd

Both standard curves are acceptable (see images below).

No amplification with either primer/probe set in the following samples:

  • 15:30-01
  • 15:30-04

I believe these are both “Control” samples (i.e. unexposed) and no amplification was expected.

All other samples amplify. See qPCR Reports for copy numbers.

RLOv Helicase Amplification & Standard Curves

WSN1 Amplification & Standard Curves

DNA Isolation & Quantification – Pinto Abalone

Isolated DNA from the following pinto abalone (Haliotis kamtschatkana) digestive gland tissues (stored in ethanol), collected by Sean Bennett as part of his Capstone project:

Accession Weight(mg)
15:30-01   194
15:30-04   67
15:31-01   34
15:31-02   107
15:31-03   83
15:31-04   80

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

Used the Roberts Lab Qubit 3.0 and the Qubit hsDNA Kit (high sensitivity). Used 1uL of template for all samples.

Samples were stored at -20C in FSH240 in the “Pinto Transcriptome DNA” box.

Results:

All samples have DNA.

Concentrations (Google Sheet): 20171226_qubit_DNA_pinto_ab

Samples Received – Pinto Abalone DNased RNA from UC-Irvine

Received DNased pinto abalone RNA from Alyssa Braciszewski at UC-Irvine. These are subset of the samples I sent her back in February.

Here’s the samples list provided by Alyssa (Google Sheet): shipment to UW of RNA samples.xlsx

The samples need to be confirmed to be free if residual RLO gDNA via qPCR. If they are clean, then will proceed to making cDNA, using provided reagents.

Reagents were stored in door of -20C in FSH 240.

Samples were stored in the provided box in the “new” -80C in FSH 235.

Sample Prep – Pinto Abalone Tissue/RNA for Collabs at UC-Irvine

We need to send half of each sample that we have from Sean Bennett’s Capstone project to Alyssa Braciszewski at UC-Irvine.

This is quite the project! There are ~75 samples, and about half of those are tissues (presumably digestive gland) stored in RNAzol RT. The remainder are RNA that has already been isolated. Additionally, tube labels are not always clear and there are duplicates. All of these factors led to this taking an entire day in order to decipher and process all the samples.

I selected samples from only those that I was confident in their identity.

I aliquoted 25μL of each RNA for shipment to Alyssa.

Tissue samples were thawed and tissue was cut in half using razor blades.

Planning to send samples on Monday.

Lisa has already assembled a master spreadsheet to try to keep track of all the samples and what they are (Google Sheet): Pinto Transcriptome

Here’s the list of samples I’ll be sending to Alyssa (Google Sheet): 20170222_pinto_abalone_samples

Here are some images to detail some of the issues I had to deal with in sample ID/selection.

 

 

 

 

 

 

 

 

 

qPCR – Withering Syndrome Phage

Ran qPCR using the following primer sets designed off of the potential WS phage sequence that Stan Langevin has sequenced:

  • 1_ORF25F_225_CSF, 1_ORF25R_399_CSF
  • 2_ORF25_121_CAB, 2_ORF25R_320_CAB
  • 3_ORF20F_121_CSF, 3_ORF20R_326_CSF

Templates tested were abalone digestive gland gDNA:

  • 06:6-41 (4/7/2008) – Positive for withering syndrome and phage; used in the MiSeq run by Stan Langevin.
  • 06:6-53 (4/9/2008) – Positive for withering syndrome and phage; used in the MiSeq run by Stan Langevin.
  • 08:4-1 – Positive for withering syndrome only – no phage.
  • UW08:22-11A – Pinto abalone naive for both withering syndrome and phage.

Master mix calcs are here: 201400820 – qPCR WS phage

All samples were run in duplicate. See qPCR Report (see Results) for plate layout, cycling params etc.

Results:
qPCR Report (PDF): Sam_2014-08-20 14-29-56_CC009827.pdf
qPCR Data File (CFX96): Sam_2014-08-20 14-29-56_CC009827.pcrd

Great news! No amplification in the 08:4-1 (positive for WS, but naive for phage)!! These results strongly suggest that these primers are specific for the WS bacteriophage! This is really cool and exciting. Next steps will be to confirm via in-situ hybridization (ISH).

Additional summary of the results:

Primer set ORF25_CSF shows the highest sensitivity.

Primer set ORF20_CSF fails to amplify anything in 06:6-53

qPCR – Withering Syndrome qPCR Assay Sample Checks

Ran qPCR on a set of water filter, fecal and tissue DNA extractions of varying copy number (based on Nate’s previous pinto abalone results) in order to get a set of high, medium and low copy number samples of each type for use in running the reproducibility aspect of the qPCR assay validation. Master mix calcs are here

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

Standard curve was the p16RK7 NcoI-linearized curve made on 20120730.

Baseline threshold was set to 400 and cycles to analyze was set to 41.

Results:

qPCR Data File (CFX96)

qPCR Report (PDF)

Here’s the list of samples analyzed and a comparison with Nate’s original data. In all instances, my results show significantly lower copy numbers (orders of magnitude lower) than Nate’s. Whether this is due to sample degradation over time is unknown. All samples have been stored at -20C since ~2005. I will discuss with Lisa and select the samples that provide us with the best range of copy numbers for use in the assay validation.