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.

 

 

 

 

 

 

 

 

 

PCR – Withering Syndrome Convetional PCR Assay Validation (Repeatibility/Reproducibility)

This is an exact repeat of the run from 20121220. Ran conventional PCR using RA5-1 and RA3-6 primers as part of assay validation for the the abalone Withering Syndrome conventional PCR. Samples were low, medium and high concentrations of WS from fecal, tissue and water DNA extractions. The positive control used was the 30,000 copy from the WS standard curve plasmid made on 20120730. Master mix calcs are here.

Cycling params:

  1. 95C – 10mins

  2. 95C – 1min

  3. 62C – 30s

Repeat Steps 2 & 3, 41 times.

Run on the Eppendorf thermalcycler.

Due to small expected product size (~160bp), samples were run on a 1.2% agarose TBE gel.

Results:

PCR – Withering Syndrome Convetional PCR Assay Validation (Repeatibility/Reproducibility)

This is an exact repeat of the run from 20121220. Ran conventional PCR using RA5-1 and RA3-6 primers as part of assay validation for the the abalone Withering Syndrome conventional PCR. Samples were low, medium and high concentrations of WS from fecal, tissue and water DNA extractions. The positive control used was the 30,000 copy from the WS standard curve plasmid made on 20120730. Master mix calcs are here.

Cycling params:

  1. 95C – 10mins

  2. 95C – 1min

  3. 62C – 30s

Repeat Steps 2 & 3, 41 times.

Run on the Eppendorf thermal cycler.

Due to small expected product size (~160bp), samples were run on a 1.2% agarose TBE gel.

Results:

PCR – Withering Syndrome Convetional PCR Assay Validation (Repeatibility/Reproducibility)

This is an exact repeat of the run from 20121220. Ran conventional PCR using RA5-1 and RA3-6 primers as part of assay validation for the the abalone Withering Syndrome conventional PCR. Samples were low, medium and high concentrations of WS from fecal, tissue and water DNA extractions. The positive control used was the 30,000 copy from the WS standard curve plasmid made on 20120730. Master mix calcs are here.

Cycling params:

  1. 95C – 10mins

  2. 95C – 1min

  3. 62C – 30s

Repeat Steps 2 & 3, 41 times.

Run on the Eppendorf thermalcycler.

Due to small expected product size (~160bp), samples were run on a 1.2% agarose TBE gel.

Results:

PCR – Withering Syndrome Convetional PCR Assay Validation (Repeatibility/Reproducibility)

This is an exact repeat of the run from 20121217. Ran conventional PCR using RA5-1 and RA3-6 primers as part of assay validation for the the abalone Withering Syndrome conventional PCR. Samples were low, medium and high concentrations of WS from fecal, tissue and water DNA extractions. The positive control used was the 30,000 copy from the WS standard curve plasmid made on 20120730. Master mix calcs are here.

Cycling params:

  1. 95C – 10mins

  2. 95C – 1min

  3. 62C – 30s

Repeat Steps 2 & 3, 41 times.

Run on the MJ thermalcycler.

Due to small expected product size (~160bp), samples were run on a 1.2% agarose TBE gel.

Results:

PCR – Withering Syndrome Conventional PCR Assay Validation (Repeatibility/Reproducibility)

Ran conventional PCR using RA5-1 and RA3-6 primers as part of assay validation for the the abalone Withering Syndrome conventional PCR. Samples were low, medium and high concentrations of WS from fecal, tissue and water DNA extractions. The positive control used was 30,000 copy from the WS standard curve made on 20120730. Master mix calcs are here.

Cycling params:

  1. 95C – 10mins

  2. 95C – 1min

  3. 62C – 30s

Repeat Steps 2 & 3, 41 times.

Run on the MJ thermalcycler.

Due to small expected product size (~160bp), samples were run on a 1.2% agarose TBE gel.

Results:

Hyperladder V was used as molecular weight markers.

qPCR – Withering Syndrome qPCR Assay Validation: Reproducibility (CFX96)

Ran additional reps of the Low and Medium Tissue samples, due to poor replication in an earlier run on these samples.

Tissue

  • Low: 09:16-18
  • Med: 09:16-22

Master mix calcs are here.

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

Results:

qPCR Data File (CFX96)

qPCR Report (PDF)

qPCR – Withering Syndrome qPCR Assay Validation: Reproducibility (CFX 96)

Ran qPCR for the reproducibility aspect of the WSN qPCR Assay Validation. Master mix calcs are here. Plate layout, cycling params, etc. can be found in the Results (see below).

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.

Samples used for “low”, “medium” and “high” copy numbers for each sample type are below, with expected fold copy number (based off of previous qPCRs):

Feces

Low: R4E 4/17/09 – 10^0

Med: R3E 7/23/09 – 10^3

High: R4E 7/23/09 – 10^4

Tissue

Low: 09:16-18 – 10^1

Med: 09:16-22 – 10^2

High: 09:20-11 – 10^5

Water

Low: 494:11-11 – 10^0

Med: 494-11-12 – 10^2

High: TAF SD A2 – 10^3

Results:

qPCR Data File (CFX96): Sam_2012-10-22 16-16-19_CC009827.pcrd

qPCR Report (PDF): Sam_2012-10-22 16-16-19_CC009827.pdf

Everything looked good except for the Low Feces sample which didn’t produce any amplification. Will identify another sample to use for the Low Feces sample.

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.

qPCR – Withering Syndrome qPCR Assay Validation: Inhibition/Repeatability – Tissue Extracts

Ran qPCR to test inhibition/repeatability using tissue extracts of high, medium and low copy numbers, also using decreasing amounts of template (5uL, 4uL, 3uL, 2uL, 1uL, 0.5uL) of each. Used the SPUD assay as an internal positive control (IPC) to assess the occurrence of inhibition in these samples. Master mix calcs are here. Plate layout, cycling params, etc can be found in the qPCR Report (see Results).

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

Results:

qPCR Data File (CFX96)

qPCR Report (PDF)

IPC comes up at cycle 19 consistently, suggesting minimal to no inhibition.