Grace’s Notebook: Qiagen RNeasy Micro Plus Kit with Geoduck hemolymph and oyster tissue

Today I also tried the Qiagen RNeasy Micro Plus Kit on geoduck (P. generosa) hemolymph (2 samples) and oyster (C. viriginica) tissue (2 samples).

What I did today:

Samples used:
img

The geoduck hemolymph (1H and 2H) are the same from what I used in my Trizol LS Reagent test. I used 250ul of each sample for the Qiagen RNeasy Extraction.

The oyster tissue samples 0120 and 0139 are from Yaamini’s DNR project. I used the whole tissue sample, and after adding the 2-ME and Buffer RLT mixture, I homogenized the samples using blue plastic mini pestles.

Protocol: page 1; page 2

I used 10ul of 2-ME per sample, with 1mL of Buffer ATL in step 1.

During steps 5-7, I centrifuged at 10,000 rcf for 30s. Tube 0139 (oyster tissue) always had some extra liquid in the spin column, but it all got spun through after the 2min centrifuge at 10,000rcf after step 7.

Eluted samples with 14ul of RNase-free water (provided in kit).

Qubit results (used 1ul of sample):

The geoduck hemolymph samples were “Out of range TOO LOW”. Here’s what the graphs looked like for both hemolymph samples:
img

The oyster tissue samples were “Out of range TOO HIGH”. Here’s what the graphs looked like for both tissue samples:
img

Next steps:

Not sure if I should spend the time to bioanalyze the oyster tissue samples from this extraction as well as from the Trizol LS Reagent Extraction

I’ll create an issue to see what next steps should be.

But it could just be that there wasn’t geoduck hemolymph in the samples I used today. I used the same sample tube for both extractions… The other one she offered had much too small of an amount of hemolymph for me to use for either extraction.

from Grace’s Lab Notebook http://bit.ly/2ReVCvP
via IFTTT

Grace’s Notebook: Trizol LS Reagent RNA extraction using Geoduck hemolymph and oyster tissue

Today I tried out the Trizol LS Reagent RNA extraction protocol on two geoduck (P. generosa) hemolymph and two oyster (C. virginica) tissue samples. (GitHub Issue #533)

What I did today:

The samples I used (from this GitHub Issue):
img

The DNR samples are the oyster tissue samples (0114 and 0116) from Yaamini’s old project.
The geoduck hemolymph samples (1H and 2H) are from when Shelly was trying things out with some geoduck that were kept here at SAFS.

I followed the same protocol as I did for the crab hemolymph, with a few modifications.

After adding the Trizol LS Reagent to the samples and centrifuging them, there was no separation into a supernatant. They all looked like this:
img

The crab samples looked like this after that same step:
img

So, instead of transfering any clear supernatant to a new tube (there wasn’t any for these samples), I just moved on and added 200ul of chloroform to each sample, vortexed, let incubate at room T for a couple mins, then centrifuged.

After the samples were left to incubate after the addition of chloroform, there was already some separation:
img

After centrifuge step with the chloroform they looked like this:
Oyster tissue:
img
Geoduck hemolymph:
img

After the addition of isopropanol, incubate at room T, and centrifuge, they all looked like this (not an obvious white gel-like pellet at the bottom… but I continued as though there were one):
img

The rest of the protocol was pretty much the same, except that I forgot to heat up the heat block earlier, so they waited at room temp for about 5 mins before they incubated at 55C.

Qubit Results (google sheet link):

There was no detectable RNA in either of the geoduck hemolymph samples.

There WAS a lot of detectable RNA in one oyster tissue sample, and too much in the other. The Qubit screen was this for the one that was too high:
img

Next move:

Try out the geoduck hemolymph and some oyster tissue samples using the Qiagen RNeasy Plus Micro Kit

Bioanalyze all samples that had detectable RNA based on Qubit results

from Grace’s Lab Notebook http://bit.ly/2S9cI2y
via IFTTT