Last Friday, Ronit quantified 1:10 dilutions of the RNA I isolated on 20181003 and the RNA he finished isolating on 20181011, but two of the samples (D11-C, T10-C) were still too concentrated.

I made 1:20 dilutions (1uL RNA in 19uL 0.1% DEPC-treated H₂O) and quantified them using the Roberts Lab Qubit 3.0, with the RNA HS assay. Used 1uL of the diluted RNA.

RESULTS

Qubit data (Google Sheet):

• 20181016_qubit_Cgigas_RNA

Everything looks good. Added final concentration values (Qubit data x 20, to account for dilution factor) to Ronit’s master sheet (Google Sheet):

• Exposure 8/29-8/30 C.Gigas

Will proceed with DNasing.

I previously isolated RNA from crab hemolymp from a lyophilized sample using TriReagent and Grace recently tried isolating RNA from crab hemolyph pellet (non-lyophilized) using TriReagent. The results for her extractions weren’t so great, so I’m giving it a shot with the following samples:

• crab 424

• crab 429

• crab 438

Isolated RNA using TriReagent, according to manufacturer’s protocol:

Added 1mL TriReagent to each tube, vortexed to mix/dissolve solute, incubated 5mins at RT, added 200uL of chloroform, vortexed 15s to mix, incubated at RT for 5mins, centrifuged 15mins, 12,000g, 4^oC, transferred aqueous phase to new tube, added 500uL isopropanol to aqueous phase, mixed, incubated at RT for 10mins, centrifuged 8mins, 12,000g, at RT, discarded supernatant, added 1mL 75% ethanol, centrifuged 5mins, 12,000g at RT, discarded supernatant and resuspended in 10uL of 0.1% DEPC-treated H₂O.

Phase separation after chloroform addition was not particularly good. Aqueous phases in sample 424 was a bit cloudy (salty?) with no defined interphase. The remaining two samples did exhibit a defined interphase and were the aqueous phases were less cloudy than sample 424, but were far from ideal.

Quantified RNA using Roberts Lab Qubit 3.0 with the Qubit RNA high sensitivity kit. Used 5uL of each sample.

RESULTS

No detectable RNA in any samples. Samples were discarded.

As has been the case for all samples in this project, RNA isolation methodologies have produced wildly inconsistent results.

Due to difficulties getting RNA from hemolymph samples stored in RNAlater, Grace is testing out lyophilizing samples before extraction. Who knows what impact this will have on RNA, but it’s worth a shot!

Isolated RNA from three crab hemolymph samples preserved in RNAlater (Test 1, Test 2, Test 3) that had been lyophilized overnight last week.

Samples were provided by Grace.

I believe the primary purpose for this particular test was to verify that the freeze dryer was a feasible tool, since Grace experienced a minor mishap when she attempted the lyohpilization initially.

Lyophilization was successful, without any mess.

TEST 3 LYOPHILIZATION

Isolated RNA using TriReagent, according to manufacturer’s protocol:

Added 1mL TriReagent to each tube, vortexed to mix/dissolve solute, incubated 5mins at RT, added 200uL of chloroform, vortexed 15s to mix, incubated at RT for 5mins, centrifuged 15mins, 12,000g, 4^oC, transferred aqueous phase to new tube, added 500uL isopropanol to aqueous phase, mixed, incubated at RT for 10mins, centrifuged 8mins, 12,000g, at RT, discarded supernatant, added 1mL 75% ethanol, centrifuged 5mins, 12,000g at RT, discarded supernatant and resuspended in 10uL of 0.1% DEPC-treated H₂O.

Quantified RNA using Roberts Lab Qubit 3.0 with the Qubit RNA high sensitivity kit. Used 5uL of each sample.

RESULTS

Qubit (Google Sheet):

• 20180912_qubit_RNA_lyophilized_crab_hemo

Only one sample (Test 3) had detectable levels of RNA (20.4ng/uL).

So, this little test demonstrates that RNA can be isolated from lyophilized samples and extracted with TriReagent. However, I have not evaluated RNA integrity on the Bioanalyzer. I think Grace has some additional samples she wanted to test this method on, so I think we’ll wait until there are more samples before we use the Bioanalyzer.

Will give sample to Grace for -80^oC storage.

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):

• 20180809_qubit_RNA_crab

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.

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.

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.

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…

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)

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

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

UPDATE 20170712: The RNA I isolated below is from incorrect regions of tissue. I misunderstood exactly what this tissue was, and admittedly, jumped the gun. The tissue is actually collected from the visceral mass – which contains gonad (a small amount) and digestive gland (a large amount). The RNA isolated below will be stored in one of the Shellfish RNA boxes and I will isolate RNA from the correct regions indicated by Grace

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.

Tissue identification is available 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).
• Reconstituted DNase I with 550μL of RNase-free H2O. Aliquoted in 100μL volumes and stored @ -20C in the “-20C Kit Components” box.

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 and quantified on the Roberts Lab Qubit 3.0 with the RNA High Sensitivity Assay (ThermoFisher Scientific) using 5μL of each sample.

Results:

Concentrations (Google Sheet): 20170710_RNA_qubit_oly_histo_blocks

Well, the good news is that there’s RNA from all the samples and it seems to be in relatively high concentrations!

The bad news is that the concentrations for 10 of the 12 samples were too high and outside the range of the Qubit RNA HS Assay! Since we don’t have the broad range RNA assay, I can’t properly quantify the remaining samples. However, these samples are being sent to Katherine Silliman at some point, so I’ll leave it up to her to quantify the samples. I’m also guessing that she’ll run them on a Bioanalyzer to assess their integrity prior to beginning library construction, so that will also yield concentrations for the samples.

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.