Submitted 3μL (~75ng) of RNA from each of the two gonad samples isolated from foot tissue embedded in paraffin histology blocks 20150408 (to assess quality of RNA) to Jesse Tsai at University of Washington Department of Environmental and Occupational Health Science Functional Genomics Laboratory:
Jesse will determine if the samples should be run on the RNA Pico or the RNA Nano chips.
Shipped (dry ice) ~15ug of total RNA from each of the following samples to Genewiz, Inc for high throughput transcriptomic sequencing (Illumina HiSeq2500, 100bp, single end).
Here’s Claire’s notebook entry on their isolation and quantification:
http://claireeolson.blogspot.com/2014/10/rna-extractions.html
Isolated RNA from the following samples provided by Jessica Blanchette (stored in RNA later):
The tocophore and veliger larval stages are neutrally bouyant (i.e. will not pellet when centrifuged). In order to separate them from the RNA Later, I used a fine mesh (don’t know mesh size; bag was labeled “Unknown”) as a “guard” between the pipette tip and the larvae. Removed RNA Later from those two groups in this fashion. However, a significant portion of the larvae in these tubes adhered to the outside of the mesh. I left the mesh “guard” in the tube, added 1mL of TriReagent and vortexed. The mesh quickly dissolved in the TriReagent, creating a milky white mix.
For the settlers samples, there was a such a large pellet already in the existing tubes, I just took ~75uL of this material, transferred to a clean tube and added 1mL of TriReagent. However, most of the debris that I transferred dissolved extremely quickly. I was expecting there to more insoluble “debris”, because marine bivalve larval shells generally don’t readily dissolve in the presence of TriReagent. So, I suspect that much of the settlers samples is not really geoduck larvae.
Due to time constraints, stored all samples O/N @-80C in TriReagent.
Samples were thawed and RNA was isolated, and DNased, using the Direct-zol RNA Miniprep Kit (ZymoResearch), eluted with 50uL of 0.1% DEPC-treated H2O, and spec’d on the NanoDrop1000.
Prior to isolation, sample V1 showed a clear phase separation that none of the other samples exhibited. Sample V1 had a pink, goopy layer on top of a clear, low-viscosity layer. All other samples retained the uniform pink coloration imparted by the TriReagent. Additionally, after addition of the EtOH in the procedure to sample V1, a large amount of white precipitate formed and settled to the bottom of the tube. This did not happen in any other samples.
Samples were stored @ -80C in “Shellfish RNA Box #5”
Results:
Overall, the yields are relatively low, as expected. Virtually all of the samples have poor OD260/280 values. Although not shown, there was a consistent shift in peak absorbance from 260nm towards 270nm, leading to the poor OD260/280 values.
Sent the following samples (in their entirety) to Cornell for Illumina HiSeq 100bp paired-end sequencing:
Apparently the Bio26 sample provided on 20140428 was incorrect. Instead, the sample should have been CF26.
Samples were initially flash frozen and then stored @ -80C (no preservatives used). No visible cells/tissue in all samples, except Bio 26. Samples were homogenized in 1mL TriReagent. Used the Direct-zol RNA MiniPrep Kit (ZymoResearch) according to the manufacturer’s protocol (including on-column DNase I procedure) for the remainder of the isolation. Eluted with 50uL of 0.1%DEPC-treated H2O and spec’d on NanoDrop1000.
Samples were stored in Shellfish RNA Box #5.
Results:
Yield and quality look great. Will pass info on to Steven and Colleen for decision on which samples to sequence.
UPDATE 20140514 – Sample sent to Cornell for Illumina RNA-seq on 20140514
Isolated RNA from the following samples (provided by Colleen Burge):
Samples were initially flash frozen and then stored @ -80C (no preservatives used). No visible cells/tissue in all samples, except Bio 26. Samples were homogenized in 1mL TriReagent. Used the Direct-zol RNA MiniPrep Kit (ZymoResearch) according to the manufacturer’s protocol (including on-column DNase I procedure) for the remainder of the isolation. Eluted with 50uL of 0.1%DEPC-treated H2O and spec’d on NanoDrop1000.
Samples were stored in Shellfish RNA Box #5.
Results:
Samples CF 3 and CF 17 likely have insufficient total RNA for sequencing at Cornell (200ng minimum required).
UPDATE 20140514 – CF2, CF34, CF35, CF70, CF71 sent to Cornell for Illumina RNA-seq on 20140514
Performed an EtOH precipitation on the sea start RNA due to some residual column resin (?) in the tubes after elution.
Added 0.1 volumes of 3M sodium acetate (pH=5.2; 10uL), 1uL glycogen (Ambion stock 5mg/mL), 2.5 volumes of ice cold 100% EtOH (275uL). Vortexed and incubated O/N at -20C.
Pelleted RNA 16,000g, 30mins @ 4C.
Discarded supe.
Washed pellet 70% EtOH.
Pelleted RNA 16,000g, 15mins @ 4C.
Repeated wash and spin.
Removed supe, resuspended RNA in 50uL of 0.1%DEPC-treated H2O and spec’d on NanoDrop1000.
Samples were stored in Shellfish RNA Box #5.
Results:
Well, overall, the RNA looks immensely better than yesterday. However, as expected, there has been some slight loss with all the additional manipulations. As such, yields are low (although, they were initially low, too). However, I think most of the samples will be usable, albeit bordering on the minimum amount of total RNA needed (200ng) at the Cornell sequencing facility…
Zymoresearch support suggested putting the samples through another set of columns to help clean up the apparent phenol carryover that was seen (absorbance peak shifted to 270nm) in the initial isolation of these samples.
Added 500uL of TriReagent to each sample and vortexed. Then, proceeded with the remainder of the protocol (excluding the DNase step). Eluted with 50uL of 0.1% DEPC-treated H2O and spec’d on NanoDrop1000.
Results:
Absolutely horrible!! I can’t even begin to fathom what has happened here. The samples run with the sample kit all worked so well; why did this whole thing have to be jacked up with the actual samples??!!
Well, I’ll do a second elution using 50uL of 0.1%DEPC-treated H2O and spec. Let’s see if that helps….
OK, I didn’t even bother spec-ing all the samples because I noticed that the elution tubes had pellets in them! When I mix the tube prior to spec-ing (which is my normal behavior), I get the top absorbance spectra that is virtually useless. When I don’t mix the samples (thus, not disturbing the pellet), I get a more “realistic” spectra, but I can’t tell if I can trust it or not. I have contacted Zymoresearch support for more help with this…
It’s tempting to simply proceed with an EtOH precipitation, but I’m a bit concerned that the pellet in the tubes is resin from the column and that it might still bind some of the RNA. However, I guess the pellet is already in the elution solution, so the RNA should be soluble and, theoretically, not be able to bind to any residual resin…
Isolated RNA from the following samples stored in RNAlater:
Spun samples 5000g, 20mins @ RT to pellet any cells. Discarded supe. Resuspended cells/debris in 1mL TriReagent. Disrupted cells by pipetting and vortexting. RNA was isolated using the Direct-zol RNA Miniprep Kit (ZymoResearch). RNA was DNase treated on-column, as described in the manufacturer’s protocol, using DNase I. RNA was eluted from the columns using 25uL of nuclease-free H2O and spec’d on a NanoDrop1000.
Results:
So, this is disheartening. Overall, the RNA looks pretty crappy; poor 260/280 ratios and a general shift in absorbance to 270nm. Plus, the yields aren’t that great. Maybe RNA left on the column and/or some sort of contaminant pushing these readings out of whack?
I will perform another elution on the columns with 50uL of nuclease-free H2O and spec that elution set:
There’s still a shift in the peak absorbance in most samples to 270nm… I’m going to combine the two sets of elutions and spec:
Although the 260/280 values are significantly better, there’s still this persistent shift of peak absorbance to 270nm. I contacted technical support for the kit and they say the absorbance shift is indicative of phenol contamination. They have advised that I add a volume of TriReagent to the RNA and re-run it through a new set of columns, following the entire RNA isolation protocol.
Isolated RNA from two samples stored in RNAlater that had either no visible pellet or a minutely visible pellet:
Samples were spun 5000g, 20mins @ RT. Supe was removed, being sure to leave behind any debris that failed to pellet. Samples were homogenized in 1mL of TriReagent by pipetting/vortexing. RNA was then isolated using the Direct-zol RNA Miniprep Kit (ZymoResearch). RNA was eluted from the column with 25uL of 0.1%DEPC-treated H2O and spec’d on a NanoDrop1000.
Results:
RNA quality looks very good, as do the yields. I’m very surprised I got anything close to 1ug out of either sample!
However, it should be noted that neither of these samples has been DNased and, as such, the yields seen above may potentially include residual gDNA carryover which would artificially inflate the yields seen above. Will DNase the samples to see how yields are affected (if at all).