Tag Archives: tanner crab

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 & Quantification – Tanner crab hemolymph

We received three Tanner crab (Chionoecetes bairdi)hemolymph samples from Pam Jensen (NOAA) yesterday. From her email to Steven:

Hi Steven,
I am sending:
tube #1 crab 3859/3656: 300 ul blood + 1300 ul RNAlater​

tube #2 crab 3665/3873: 300 ul blood + 1300 ul RNAlater
​tube #3 crab 3665/3873: 200 ul blood + 1400 ul RNAlater​

The tubes hold max of 1600 ul. Will know on Sun or Mon if either crab is infected w Hematodinium.

Tracking info to follow.
Pam

Samples were stored at 4C O/N.

Here’s what the samples looked like before processing:

The samples are extremely cloudy. I’m not sure if this is expected.

Processed samples using RNAzol RT (MRC) according to the manufacturer’s protocol for Total RNA Isolation.

Pelleted samples at 5000g for 5 mins and the samples looked like this:

Decided to pellet samples for an additional 10mins. The pellet was more compact. Transferred supernatant to clean tube, since it seemed to contain “debris” (maybe cells?). Processed pellet with RNAzol RT. Brief rundown of procedure (all steps at room temp):

  1. Transferred supe to clean tube.
  2. Added 1mL RNAzol RT to pellet and mixed by repeated pipetting (solution was cloudy and slightly viscous).
  3. Added 400uL of 0.1% DEPC-treated H2O and mixed vigorously by hand.
  4. Incubated for 10mins.
  5. Centrifuged 12,000g for 15mins.

    Samples looked like this:

    This is not normal. Usually the supernatant is the clear portion, while the blue layer is below that.
  6. Transferred 750uL of the clear portion to clean 1.7mL tube.

  7. Added equal volume of isopropanol, mixed by inversion. Appeared to be a very high amount of genomic DNA precipitation visible in the tube.
  8. Incubated for 10mins.
  9. Centrifuged 12,000g, 15mins.

    Samples looked like this:

    It appears that the nucleotides (the white interphase) are suspended on a “cushion” of higher density solution, instead of pelleted at the bottom of the tube.
  10. Removed/discarded higher density solution, leaving the white layer on the bottom of the tube.

  11. Centrifuged 12,000g, 15mins.
  12. Discarded supe.
  13. Washed pellet with 75% ethanol.
  14. Centrifuged 8,000g, 3mins.
  15. Repeated Steps 12, 13, & 14, 1x.
  16. Discarded ethanol.
  17. Resuspended RNA in 50uL 0.1% DEPC-treated H2O. Pellets did not solubilize on their own. I dispersed the pellets by repeated pipetting (P200). Remaining insoluble material was pelleted (12,000g, 30s) and supernatant was transferred to a new 1.6mL tube.

RNA was quantified using the Qubit 3.0 and the Qubit HS RNA Assay. Used 5uL of each sample.

Results:

20171107_qubit_tanner_crab_hemo (Google Sheet)

Sample ID Conc. (ng/uL) Total Yield (ng)
3859/3656 0.44 22
3665/3873 1.66 83
3665/3873 2.04 102

Interestingly, both samples from the same crab had similar/decent yields.

Samples were labeled and stored at -80C in Shellfish RNA Box #6

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