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
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.
Isolated RNA from pinto abalone larvae that were sampled on 20110930. Samples were provided by Liza.
Due to large volumes of sea water in each sample (ranging from 500uL to 2000uL), samples had to be thawed entirely and then as much sea water was removed as physically possible. Samples were homogenized in 500uL of TriReagent (Molecular Research Center). An additional 200uL of TriReagent was added to each sample after homogenization to bring the final sample volume to ~1000uL. Samples were then processed according to the manufacturer’s protocol. Samples were resuspended in 50uL of 0.1% DEPC-treated H2O, heated at 55C for 10mins to coax the RNA into solution and spec’d.
Yields seem excellent (~80ug of total RNA per sample) as do the OD260/280 ratios. RNA will be stored @ -80C (will pass samples to Liza to decide on the physical location in the -80C) until a decision is made on what to do with the samples next.