Tag Archives: RNA

RNA Isolation & Quantificaiton – Tanner Crab Hemolymph

Isolated RNA from 40 Tanner crab hemolymph samples selected by Grace with the RNeasy Plus Micro Kit (Qiagen) according to the manufacturer’s protocol, with the following modifications:

  • Added mercaptoethanol (2-ME) to Buffer RLT Plus.

  • All spins were at 21,130g

  • Did not add RNA carrier

  • Used QIAshredder columns to aid in homogenization and removal of insoluble material

  • Eluted with 14uL

RNA was quantified using the Qubit RNA HS (high sensitivity) Assay and run on the Roberts Lab Qubit 3.0.

Used 1uL of sample for quantification.

RNA was returned to the -80C box from where original samples had been stored (Rack 2, Row 3, Column 4).


RESULTS

Qubit quantification (Google Sheet):

Overall, the results aren’t great. Only 15 samples (out of 40) had detectable amounts of RNA. Yields from those 15 samples ranged from 40ng – 300ng, with most landing between 50 – 100ng.

Will pass info along to Grace. Will likely meet with her and Steven to discuss plan on how to move forward.

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Bioanalyzer – Tanner Crab RNA Isolated with RNeasy Plus Mini Kit

Ran the four Tanner crab RNA samples that I isolated yesterday on the Seeb Lab Bioanalyzer 2100 (Agilent) using the RNA Pico 6000 Kit.

Samples were run following kit protocol:

  • Chip priming station in Position C with syringe clip at top position

  • RNA denatured at 70C for 2mins and stored on ice.

  • RNA ladder aliquot was from 20160826 by Hollie Putnam.


RESULTS

Bioanalyzer data file (XAD):

ELECTROPHEROGRAMS:


GEL REPRESENATATIONS


These results look great to me. Clear, defined peaks/bands, representing ribosomal RNA.

Oddly, one sample (crab_506) appears to be shifted, relative to the other three, despite exhibiting the same peak/banding pattern. Not sure what would cause something like this; contaminants?

Regardless, we finally have clean RNA and have a usable Bioanalyzer profile to use for reference for crab RNA.

NOTE: The lanes marked with red on the gel representation image indicate that a ribosomal integrity number (RIN) could not be calculated. This is to be expected! The RIN is based on the expectation of two rRNA bands. The anomaly is sample crab_451 – a RIN was actually determined for that sample!

Will likely move forward with additional RNA isolations using the RNeasy Plus Kit (Qiagen).

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RNA Cleanup – Tanner Crab RNA

In a continued attempt to figure out what we can do about the tanner crab RNA, Steven tasked me with using an RNeasy Kit to cleanup some existing RNA.

Here’re the samples grace provided:


All of the RNA had some sort of undissolved/insoluble material present. Here’s an example (this is the worts of the bunch – others did not have such large/dense pellets):


Samples were cleaned up using the [RNeasy Plus Mini Kit (Qiagen)]. Added 350uL of Buffer RLT Plus (no beta-mercaptoethanol added) to each sample, vortexed, and then processed according to the manufacturer’s protocol (skipped gDNA Eliminator spin column step).

Samples were eluted with 30uL of nuclease-free water.

Samples were quantified using the Roberts Lab Qubit 3.0 with the RNA High Sensitivity asssay (Invitrogen). Used 5uL of sample for measurements.

Samples were also assessed with the Roberts Lab NandoDrop1000.

Samples were recovered from the pedestal after measurement.

RNA was given to Grace for storage at -80C.


RESULTS

Qubit measurements (Google Sheet):
20180731_qubit_RNA_crab_cleanup


NanoDrop Table:


All concentrations were too low for detection via NanoDrop.

Qubit quantification indicate yields ranging from ~25ng to ~192.5ng.

Will share info with Grace and let her compare these numbers to her original concentrations to see if there’s any differences.

Regardless, based on my earlier RNA isolation today, these samples should now be much cleaner and we should be able to trust the Qubit quantifications.

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RNA Isolation – Tanner Crab Hemolymph Using RNeasy Plus Mini Kit

Tanner crab RNA has proved a bit troublesome. As such, Steven asked me to try isolating some RNA using the RNeasy Plus Mini Kit (Qiagen) to see how things would turn out.

Grace provided me with the following samples:


Crab hemolymph had been collected (100uL?) and preserved with 1mL (?) of RNAlater. Grace pelleted the samples, removed the supernatant, and stored the pelleted material at -80C. Here’s what that looked like:


RNA was isolated according to the manufacturer’s protocol – following guideline for samples with < 1 x 106 cells.

One interesting thing that happened is a precipitate formed after adding the initial buffer to the sample:

A solid precipitate formed in each of the tubes that could not be dispersed – it actually looked like a small piece of paper was now present in each tube.

Samples were spun and the supernatant was utilized (this was the normal progression of the protocol, regardless of this precipitate forming).

Samples were eluted with 30uL of nuclease-free water.

Samples were quantified using the Roberts Lab Qubit 3.0 with the RNA High Sensitivity asssay (Invitrogen). Used 5uL of sample for measurements.

Samples were also assessed with the Roberts Lab NandoDrop1000. Samples were recovered from the pedestal after measurement.

RNA was given to Grace for storage at -80C.


RESULTS

Qubit measurements (Google Sheet):
20180731_qubit_RNA_crab_isos


NanoDrop Spec Curves:


NanoDrop Table:


Overall, the isolation looks pretty good. The purity looks good (NanoDrop 260/280 ratios) and the absorbance peak at 260nm is exactly where we would want/expect it to be.

The yields (according to the Qubit) are OK. They range from ~37ng – 350ng.

The important part is that this method produced clean RNA, which means the quantification is believable. I think Grace’s earlier RNA isolations using RNAzol RT had too much contamination carried over, leading to incorrect quantification measurements.

Going forward, I think we need to use some sort of isolation kit, however, we will be testing out good, old TriReagent as well.

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RNA Cleanup – Tanner Crab RNA Pools

Grace had previously pooled a set of crab RNA in preparation for RNAseq. Yesterday, we/she concentrated the samples and then quantified them. Unfortunately, Qubit results were not good (concentrations were far below the expected 20ng/uL) and the NanoDrop1000 results yielded awful looking curves.

In an attempt to figure out what was wrong, I decided to use the RNeasy Plus Mini Kit (Qiagen) on the three pools. I did this due to the poor spec curves seen in the NanoDrop1000 measurements. Additionally, all of the RNA pools had undissolved/insoluble bits floating around in them. My thinking was that excess contaminants/salts could be interfering with the Qubit assay. Removing these could/should enlighten us as to what the issue might be.

Followed the manufacturer’s protocol for RNeasy MiniElute Cleanup Kit (as the RNeasy Plus Mini Kit uses the same reagents/columns for RNA purification) for samples with <100uL.

Samples were quantified on the RobertsLab NanoDrop1000 (ThermoFisher) and the Qubit 3.0 (ThermoFisher) using the RNA high sensitivity (HS) Kit. Used 1uL of each sample.

Results:

Qubit (Google Sheet): 20180719_qubit_RNA_crab_pools

NanoDrop:

The NanoDrop did not detect any RNA in the samples.

The Qubit did not detect any RNA in Crab Pool 1. The other two samples had similar concentrations (~7ng/uL). This would mean a total of ~84ng of RNA was present in each of those two samples.

All pools were expected to have well over 1000ng of RNA.

Will have to think about what should be done, but I would lean towards attempting to run some “test” samples through the RNeasy Cleanup kit to see if that would help get us more accurate Qubit readings? I don’t know, though…

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RNA Isolation – Olympia oyster gonad tissue in paraffin histology blocks

My previous go at this was a little premature – I didn’t wait for Laura to fully annotate her slides/blocks. Little did I know, the tissue was mostly visceral mass and, as such, I didn’t hit much in the way of actual gonad tissue. So, I’m redoing this, now that Grace has gone through and annotated the blocks to point out gonad tissue. SN-10-16 was sent to Katherine Silliman on 20170720.

Isolated RNA from Olympia oyster gonad previously preserved with the PAXgene Tissue Fixative and Stabilizer and then embedded in paraffin blocks. See Laura’s notebook for full details on samples and preservation.

 

RNA was isolated from the following samples using the PAXgene Tissue RNA Kit (Qiagen). Gouged samples from the blocks weighing ~10mg from each of the tissues and processed according the protocol for isolating RNA from blocks of paraffin-embedded tissues.

Background on all of this is in this GitHub Issue

NF-10-22
NF-10-23
NF-10-24
NF-10-26
NF-10-28
NF-10-30
SN-10-16
SN-10-17
SN-10-20
SN-10-25
SN-10-26
SN-10-31

IMPORTANT:

  • Prior to beginning, I prepared an aliquot of Buffer TR1 by adding 40μL of β-mercaptoethanol (β-ME) to 4000μL of Buffer TR1)

Isolated RNA according to the PAXgene Tissue RNA Kit protocol with the following alterations:

  • “Max speed” spins were performed at 20,000g.
  • Tissue disruption was performed by adding ~25-50 glass beads (425 – 600μm diameter) with the Disruptor Genie @ 45C for 15mins (in the Friedman Lab).
  • Shaking incubation step was performed with Disruptor Genie
  • Samples were eluted with 27μL of Buffer TR4 x 2, incubated @ 65C for 5mins, immediately placed on ice.

 

Results:

Samples were not quantified due to lack of proper RNA Qubit assay AND the computer that our NanoDrop1000 is hooked up to is dead. Will have Katherine Silliman perform quantification.

Samples were stored at -80C temporarily.

Samples will be sent to Katherine Silliman for high-throughput library construction and sequencing once I hear back from her regarding her availability to receive the samples.

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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

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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.

 

 

 

 

 

 

 

 

 

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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
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