Tracked down and submitted four cassettes containing pinto abalone digestive gland tissue that Sean Bennett prepared, as part of his Capstone project. A full list of samples from his project is here (Google Sheet): Pinto Transcriptome
Sent them to Diagnostic Pathology Medical Group for histology processing.
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
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
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.
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.
Pooled equal quantities (10ug) from each of the four samples in each treatment, for a total of 40ug of total RNA in each pooled set of total RNA. Samples were submitted to HTGU for Illumina sequencing, 36bp, single-end reads.