Tag Archives: RNA quantification

RNA Isolation – Geoduck Gonad in Paraffin Histology Blocks

Isolated RNA from geoduck gonad previously preserved with the PAXgene Tissue Fixative and Stabilizer and then embedded in paraffin blocks. See Grace’s notebook for full details on samples and preservation.

 

RNA was isolated from only two samples using the PAXgene Tissue RNA Kit (Qiagen) from the following geoduck sample blocks to test out the kit:

  • 34
  • 42

IMPORTANT:

  • Prior to beginning, I prepared Buffer TR1 by adding 10μL of β-mercaptoethanol (β-ME) to 1000μL of Buffer TR1). This will be good for up to six weeks at RT.
  • Reconstituted DNase I with 550μL of RNase-free H2O. Aliquoted in 100μL volumes and stored @ -20C in the “-20C Kit Components” box.

Five 5μm sections were taken from each block.

Isolated RNA according to the PAXgene Tissue RNA Kit protocol with the following alterations:

  • “Max speed” spins were performed at 19,000g.
  • Tissue disruption was performed with the Disruptor Genie @ 45C for 15mins.
  • Samples were eluted with 34μL of Buffer TR4, incubated @ 65C for 5mins, immediately placed on ice and quantified on the Roberts Lab NanoDrop1000.

Samples were stored at -80C in Shellfish RNA Box #5.

NOTE: The spreadsheet linked indicates other samples exist in the slots that I placed these two samples. Will need to update the spreadsheet to be accurate.

Results:

 

 

Looks like the kit worked! Yields are pretty good (~800ng) from each. The 260/280 ratios are great for both samples. Oddly, the 260/230 ratios for the two samples are pretty much polar opposites of each other; not sure why.

Will proceed with the remainder of the samples that were selected by Steven and Brent. Or, maybe I should try to make some cDNA from these RNA samples to verify the integrity of the RNA…

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RNA Isolation – Jessica’s Geoduck Larval Stages

Isolated RNA from the following samples provided by Jessica Blanchette (stored in RNA later):

  • Trocophore 1 (T1)
  • Trocophore 2 (T2)
  • Veliger 1 (V1)
  • Veliger 2 (V2)
  • Settlers Interphase 1 (S1)
  • Settlers Interphase 2 (S2)

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.

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RNA Isolation – Colleen Sea Star (Pycnopodia) Coelomycete Sample

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

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RNA Isolation – Colleen Sea Star (Pycnopodia) Coelomycete Samples

Isolated RNA from the following samples (provided by Colleen Burge):

  • Bio 26 (a LARGE amount of tissue/debris in this sample!)
  • CF 2
  • CF 3
  • CF 17
  • CF 34
  • CF 35
  • CF 70
  • CF 71

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

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Ethanol Precipitation – Colleen’s Sea Star Coelomycete RNA from Yesterday

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…

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RNA Clean Up – Colleen’s Sea Star Coelomycete RNA from 20140416

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…

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RNA Isolation – Colleens’ Sea Star Coelomycetes Samples

Isolated RNA from the following samples stored in RNAlater:

  • TH52 3.28.14 c-fluid
  • TH54 3.28.14 c-fluid
  • CH55 3.28.14 c-fluid
  • CH56 3.28.14 c-fluid
  • CH57 3.28.14 c-fluid
  • TH65 3.28.14 c-fluid
  • TH66 3.28.14 c-fluid
  • TH67 3.28.14 c-fluid

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.

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RNA Isolation – Sea Star Coelomocytes (from Colleen)

Isolated RNA from two samples stored in RNAlater that had either no visible pellet or a minutely visible pellet:

  • Control P26
  • Filt. Inj. P8

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).

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RNA Isolation – Sea Star Coelomocytes (provided by Colleen Burge)

Tried another method of RNA Isolation for comparison with regular TriReagent method.

Used the Direct-zol RNA MiniPrep Kit (Zymo Research) on the following samples stored in RNAlater:

  • P6 Control
  • P16 Filt. Inj.

Pelleted samples in RNAlater by spinning 5000g, 10mins @ RT. Removed RNAlater, lysed pellets in 1mL TriReagent. Split each sample equally into two tubes (500uL in each tube). Added equal volumes of 100% ethanol to each tube and vortexed. Transferred samples to spin columns and followe manufacturer’s protocol. Eluted with 25uL of nuclease-free H2O (provided in kit). Spec’d on NanoDrop1000.

Results:

RNA quality is very good (based on 260/280 ratios). This turned out much better than the previous attmpt using the basic TriReagent method. However, the previous attempt (see 20140401) may have been compromised by me being too aggressive when collecting the aqueous phase. Knowing how little sample was present, I may have been overzealous in trying to gather too much of the aqueous phase, leading to the phenol carryover that was evident.

Regardless, these columns seem to do an excellent job of eliminating even salt carryover, as we frequently see high absorbance at 230nm with marine samples; particularly those stored in RNAlater.

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RNA Isolation – Sea Star Coelomocytes (provided by Colleen Burge)

Isolated RNA from the following samples (stored in RNAlater):

P18 Control 3/17/14

P10 Filt. Inj. 3/17/14

These were “trial” RNA isolation runs to determine what yields we could expect from samples of this nature.

Both samples had very small tissue/cell pellets. Tubes were spun @ 5000g for 10mins at RT to ensure all cells were pelleted. RNAlater was removed and pellets were lysed using 1000uL of TriReagent, supplemented with 8uL of PolyAcryl carrier. PolyAcryl Carrier was used to enhance RNA recovery from such small starting materials. Remainder of procedure followed manufacturer’s protocol. RNA was resuspended in 20uL of 0.1% DEPC-H2O and spec’d on a NandoDrop1000.

Results:

As can be seen by the absorbance spectrum plots (top image), there is clear phenol contamination (indicated by shift of absorbance peak to 270nm, instead of the peak being at 260nm). Additionally, there’re large peaks at 230nm in each of the two samples, suggesting other contamination (high residual salts, ethanol?). Additionally, the 260/280 ratios are subpar for RNA quality (i.e. <1.9). However, these ratios could be skewed by the the residual phenol present in both samples. I may perform an ethanol precipitation on these just to see if I can get them cleaned up.

Yields for both samples are very promising.

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