RNA Isolation – Abalone Water Filters for RLO Viability

Water filters stored at -80C in ~1mL of RNAzol RT were provided by Lisa. This is part of an experiment (and Capstone project) to assess RLO viability outside of the host.

The samples were thawed and briefly homogenized (as best I could) with a disposable plastic pestle. The samples were then processed according to the manufacturer’s protocol for total RNA isolation. Samples were resuspended in 25μL of nuclease-free water (Promega).

Immediately proceeded to DNase treatment.

The experimental samples and the various treatments are viewable in the “Viability Trial 2″ tab of Lisa’s spreadsheet (Google Sheet): RLO Viability & ID50

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.

 

 

 

 

 

 

 

 

 

DNase Treatment – Water Filter RNA from 20161130

Continued preparation of this RNA to assess withering syndrome viability in the water column. I treated the RNA I isolated on 20161130 using the Turbo DNA-free (Ambion) DNase kit, according to their protocol.

Added the following to each sample:

  • 2.5μL 10x buffer
  • 1.5μL H2O
  • 1μL DNase

Incubated @ 37C for 1hr.

Added 0.1 volumes (2.5μL) of DNase Inactivation reagent and incubated at RT for 2mins (with mixing). Pelleted inactivation reagent: 10,000g, 2mins, RT. Transferred supe to new tube.

Samples were labelled as “DNased RNA”, their existing sample name (see below), and stored @ -80C.

Sample names:

  • T0A
  • T0B
  • T1A
  • T1B
  • T1A C
  • T1B C
  • T3A
  • T3B
  • T7A
  • T7B
  • T7A C
  • T7B C

RNA Isolation – Abalone Water Filters

Lisa provided me with a set of water filters stored in 1mL RNAzol RT @ -80C to isolate RNA from. This is an attempt to assess withering syndrome viability from within the water.

The samples were thawed and briefly homogenized (as best I could) with a disposable plastic pestle. The samples were then processed according to the manufacturer’s protocol for total RNA isolation. Samples were resuspended in 20μL of 0.1%-DEPC H2O.

Samples were stored @ -80C. Will DNase next week.

The sample names are as follows (the ‘C’ is short for “Control”, per Lisa):

  • T0A
  • T0B
  • T1A
  • T1A C
  • T1B C
  • T3A
  • T3B
  • T7A
  • T7B
  • T7A C
  • T7B C

 

DNase – Withering Syndrome RNA

Withering syndrome RNA, extracted from water filters, was confirmed via qPCR to contain detectable quantities of withering syndrome DNA. Before proceeding to make cDNA, residual gDNA carryover from the RNA extraction needs to be removed.

Performed DNase treatment with the Turbo DNA-free Kit (Ambion/Life Technologies), following the rigorous. Used 10μL of each of the following template RNA:

  • Day 0-1
  • Day 3-1
  • Day 7-1
  • Day 11-1

Reactions were performed in a volume of 50μL. Used 1μL of DNase for the first 30mins @ 37C and then added an additional 1μL of DNase for the final 30mins @ 37C. Samples were inactivated according to the manufacturer’s protocol and spec’d on the Roberts Lab NanoDrop1000.

Results:

Yields are consistent. The OD260/280 values are still poor (didn’t expect them to change, though). Will qPCR to verify removal of gDNA.

qPCR – Withering Syndrome RNA Check

Ran withering syndrome qPCR on the following RNA samples provided by Lisa to determine if it’s necessary to DNase treat them prior to performing reverse transcription:

I believe these were extracted from water filters collected from basement abalone tank water. The OD260/280 ratios are pretty bad, particularly for RNA, but will proceed anyway.

Master mix calcs are here: 20150318 – qPCR WSN RNA Test

Since I’m anticipating using 1000ng of RNA in the reverse transcription reactions (25μL reaction volume = 40ng/μL), I only used 1μL of RNA (~100ng), and positive control, template in each reaction to semi-accurately represent the quantity of RNA that would be loaded in a qPCR reaction with 2μL of cDNA.

All samples were run in duplicate. Positive control was the 3e6 p18RK7 standard curve sample from 20120731.

Results:

qPCR Report (PDF): Sam_2015-03-18 10-11-35_CC009827.pdf
qPCR Data File (CFX96): Sam_2015-03-18 10-11-35_CC009827.pcrd

All samples are positive for withering syndrome. Will proceed with DNase treatment.

RNA Isolation – Pinto Abalone Larvae from 20110930

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.

Results:

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.

RNA Isolation & DNase – Abalone Dg

Isolated RNA from Red Abalone Dg (10:25-24, 25, 26) using TriReagent, according to protocol. These samples are expected to have high levels of the Rickettsia phage that I’ve been attempting to investigate. The tissue samples were taken on 20110224 by Lisa and stored in RNA Later @ 4C. Remaining tissue will be returned to existing tubes and stored @ -80C. Previously, RNA from Ab Dg had been isolated using the MoBio RNA PowerSoil Kit due to its ability to produce extremely clean RNA from Dg (a nasty tissue). However, we currently do not have that kit. RNA was resuspended in 100uL of 0.1% DEPC-H2O and spec’d on the Roberts Lab ND1000. RNA will be stored in “Sam’s -80C Box #1″ in the Roberts’ Lab -80C freezer.

Results:

As expected, the RNA quality is low (based on OD260/280), as the TriReagent method of isolation does not clean up the RNA as extensively as the MoBio RNA PowerSoil Kit which has been previously used. Additionally, the RNA solution is brown colored. Will DNase RNA.

 

DNase – Abalone Dg RNA (from earlier today)

DNased Abalone Dg RNA that was isolated earlier today using Ambion’s Turbo DNA-free Kit following the “rigorous” protocol. Briefly, 5ug of RNA was brought up to 45uL with nuclease-free H2O. Reaction was brought to 50uL with 10x DNase Buffer and 1uL of DNase. Sample was incubated @ 37C for 30mins. An additional 1uL of DNase was added, mixed and incubated for 30mins. DNase was inactivated, supe transferred to clean tube and spec’d on Roberts’ Lab ND1000. DNased RNA will be stored in “Sam’s RNA Box #1″ in the Roberts’ Lab -80C freezer.

Results:

OD260/280 values still look poor (which was expected based on initial isolation from earlier today). Will check DNased RNA for residual gDNA carryover.