Tag Archives: hemolymph

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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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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Received Hard Clam Samples from Rutgers

30 gill tissue samples in RNA Later from CA, MA, & MAX each.

30 hemolymph samples (in RNA Later?) from CA, MA, & MAX each.

Presumably these are from the same individuals. Tubes were boxed (a total of 3 boxes), labeled and stored @ -80C.

Here is a note included from Emily with the samples .

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Package – Hard Clam Samples from Rutgers

Rec’d package of hard clam samples from Emily @ Rutgers on wet ice. Package contained numerous 1.5mL snap cap tubes separated in to groups in zip lock bags. Stored temporarily @ 4C. Will catalog and then store @ -80C.

Three documents included with package:

Note from Emily

Sample info, pg. #1

Sample info, pg. #2

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Samples Received – Hard Clam samples from Rutgers and MBL

 *Important Note: These were received while I was out of lab. This notebook entry was added 20101021*

Received sets of gill tissue and hemolymph in RNA Later from Rutgers (Emily). Here’s the note that was included with the samples.

Received set of gill tissue in RNA Later MBL (Scott Lindell).

All samples were stored @ -80C.

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Package – Hard Clam gill tissue/hemolymph in RNA later

Rec’d package from Rutgers (Emily Pearson) containing two large Ziplock bags on “wet” ice, each of those containing smaller bags with sample tubes in them. One large bag contains gill tissue samples and the other large bag contains hemolymph samples. Samples will temporarily be stored @ 4C until they can be catalogued and boxed by Lexie later today.

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Bleeding – Hard Clams

Bled 7 clams from 20090108 and 20090109. Bled clams using a 23g 1.5 needle on a 3mL syringe. Fluid was gathered and ranged from ~0.4-1.0mL. Hemolymph was transferred to individual 1.5mL snap cap tubes and spun @ 100g for 30mins @ 4C. Most of the supe was removed, but left ~100uL in each tube to avoid disturbing any pellet. Samples were stored @ -80C in the red box with previous hard clam hemo samples.

NOTE: One sample was EXTREMELY cloudy. Likely not hemos.

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RNA – Precipitation continued from yesterday

Transferred supe to a fresh tube and added 1mL 70% EtOH to remaining pellet. Spun samples max speed @ 4C 30 mins. Removed supe and washed pellets with 1mL 70% EtOH. Spun max speed 10 mins. Removed supe . Resuspended the “supe” sample in 50uL 0.1%DEPC-H2O and the “pellet” sample in 100uL 0.1%DEPC-H2O.

Results: 260/280 ratios look good. The 260/230 ratios are still horrible. Total yield from these two samples are ~5ug. Will get more hemolymph from clams in order to use more total RNA in the mRNA isolation to maximize cost saving.

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RNA – Reprecipitation of hard clam RNA from yesterday

Because of the relatively large size of the pellets vs. the amount of RNA, I think another round of precipitation would be best to help remove additional residual salt carryover. Will precipitate O/N according to Ambion PolyA Purist protocol. RNA pellets were resuspended in 250uL of 0.1%DEPC-H2O and precipitated O/N @ -20C.

NOTE: Upon adding 100% EtOH to sample, the solution turned very cloudy and a white precipitate immediately formed inside the tube. I do not think this precipitate is RNA. Tomorrow, before spinning the tube, I will transfer the supe to a fresh tube and process both tubes simultaneously. Hopefully this will remove/eliminate most of the excess salt or whatever seems to be forming the pellet.

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