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

DNA Isolation – Claire’s C.gigas Female Gonad

Trying this sample again(!!), but will now use TE for pellet resuspension to prevent sample degradation. Incubated sample RT on rotator in 500uL of DNazol + 2.7uL of Proteinase K (Fermentas; Stock 18.5mg/mL) for 5hrs. Added additional 500uL of DNazol, mixed gently and followed DNazol manufacturer’s protocol. Performed first pellet was with 70% DNazol/ 30% EtOH solution. Resuspended pellet in 200uL of TE and spec’d on NanoDrop1000.

Results:

Yield is good. 260/280 value is good. 260/230 value is poor. Will run on gel to evaluate integrity.

Loaded 10uL (~830ng) on 1.0% agarose 1x modified TAE gel stained with EtBr.

Gel Loading Guide:

Lane 1 – Hyperladder I (Bioline)

Lane 2 – C.gigas female gonad gDNA (CgF)

Well, this certainly looks much better than previous preparations, in that there is an obvious high molecular weight band present (previously, this had been absent). The low molecular weight bands/smears are possibly RNA carryover and/or degraded DNA. Will discuss with Steven and then, most likely, bring downtown for Illumina sequencing.

UPDATE 20140508: Downtown sequencing facility says there’s only ~800ng of DNA! This is a far cry from the minimum amount needed for sequencing (6ug). Looking at the gel above and comparing sample band intensity to the ladder band intensities suggests that the downtown sequencing facility is correct. I loaded 10uL of DNA on the gel and the intensity of the high molecular weight band is similar to the 400bp band intensity. This corresponds to 40ng of DNA. That means the CgF gDNA band is 40ng/10uL = 4ng/uL. I resuspended the gDNA pellet in 200uL of TE, so 200uL x 4ng/uL = 800ng; exactly what the sequencing facility says they measured…

I’m not entirely sure what is happening here. Until very recently, there were almost never such egregious differences between the NanoDrop measurements and what they were measuring downtown at the sequencing facility. It seems as though they have changed the way they quantify samples (possibly using an Agilent Bioanalyzer instead of the Life Technologies Qubit fluorometer?), but this doesn’t mean their measurements are incorrect. However, I’m starting to suspect that the reason the initial sequencing of this sample was due to an overestimation of the quantity of input DNA (since I believe they were still using the fluorometer back then).

As such, it’s become clear that C.gigas gonad samples seem to yield poor quantities of gDNA, relative to the amount of input material. Additionally, there may be insufficient sample left to generate a useable quantity of gDNA to complete this resequencing effort.

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…

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…

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.

DNA Isolation – Test Sample

Due to the recent poor quality gDNA that has been isolated from C.gigas gonad, I decided to do a quick test using TE for DNA pellet resuspension in hopes that old Buffer EB (Qiagen) or old nuclease-free H2O (Promega) are to blame for the apparent, rapid degradation that I’ve experienced.

Isolated gDNA from a C.gigas female gonad sample (EV2 141 go) provided by Mac. Isolated gDNA using DNazol (Molecular Research Center):

  1. Incubated ~25mg of tissue O/N @ RT in 500uL of DNazol + 100ug/mL Proteniase K (2.7uL of 18.5mg/mL Fermentas stock) on rotator.

  2. Added additional 500uL of DNazol and briefly disrupted remaining tissue with a few pipette strokes.

  3. Pelleted debris by spinning 10mins, 10,000g @ RT.

  4. Transferred supe to new tube and repeated Steps 3 & 4 one time.

  5. Added 500uL of 100% EtOH; mixed by inversion.

NOTE: Despite initial appearance of white cloudy appearance after EtOH addition, cloudiness dispersed upon inversion and no visible DNA strands were present

  1. Pelleted DNA by spinning 5000g 5mins @ RT.

  2. Removed supe and washed pellet with 1mL of a 70% DNazol+30% EtOH solution.

  3. Removed supe and washed pellet with 1mL 70% EtOH.

  4. Repeated Step 8 two times.

  5. Discarded supe, quick spun tube to pool residual EtOH. Removed all residual EtOH.

  6. Resuspended in 200uL of TE (pH = 8.0) and incubated at RT for 5mins.

  7. Pelleted insoluble material 12,000g 10mins @ RT.

  8. Transferred supe to clean tube.

  9. Spec’d on NanoDrop1000.

  10. Ran ~500ng on 1.0% agaroase 1x modified TAE gel to evaluate integrity.

Results:

260/280 value looks excellent, but, as always seems to be the case with DNazol/TriReagent, the 260/230 value looks crappy. Will investigate gDNA integrity on agarose gel.

Gel Loading:

Lane 1 – Hyperladder I (Bioline)

Lane 2 – EV2 141 go C.gigas female gonad gDNA

Well, look at that! A nice, clear, high molecular weight band! It looks like my Buffer EB and/or nuclease-free water are is contaminated. Have discarded both. Will re-isolated Claire and Mac’s gDNA.

Phenol-Chloroform DNA Clean Up – Mac and Claire’s Samples (from 20140410)

Due to low 260/230 values and Mac’s smeary sample, performed a phenol-chloroform DNA cleanup on the samples isolated 20140410.

  1. Brought volume of each sample to 200uL with Buffer EB (Qiagen).

  2. Added an equal volume (200uL) of 25:24:1 Phenol/Chloroform:Isoamyl alcohol.

  3. Mixed on rotator for 20mins @ RT.

  4. Separated aqueous/organic phases by spinning at 12,000g 5mins @ RT.

  5. Transferred aqueous phase to new tube. Repeated steps 2-4 until samples exhibited no more interphase. Combined aqueous phases in to a single tube for each of the two samples.

  6. Added and equal volume of chloroform (170uL).

  7. Mixed on rotator for 20mins @ RT.

  8. Separated aqueous/organic phases by spinning at 12,000g 5mins @ RT.

  9. Transferred aqueous phase to new tube.

Performed an ethanol precipitation on each sample.

  1. Added 0.1 volumes of 5M sodium acetate (pH = 5.2).

  2. Added 2 volumes of ice cold 100% EtOH.

  3. Incubated 20mins @ -20C.

  4. Pelleted DNA by spinning 16,000g, 20mins @ 4C.

  5. Discarded supe and washed pellets with 1mL 70% EtOH.

  6. Pelleted DNA by spinning 16,000g, 5mins @ 4C.

  7. Repeated steps 5-6 one time.

  8. Removed all supernatant and resuspended in 100uL of nuclease-free H2O.

  9. Spec’d on NanoDrop1000.

NOTE: Mac’s sample exhibited the same chunky/cloudiness upon addition of 100% EtOH that has been seen previously by both her and myself…

Results:

So, the clean up seemed to work wonders on the 260/230 values. Not surprisingly, Mac’s sample didn’t clean up nearly as nicely as Claire’s, based on my observations of the odd behavior during EtOH precipitation.

And, despite the nice, clean looking peaks, the 260/280 ratios are actually WORSE than the original isolation. Will run on gel for a further assessment of quality/integrity.

Loaded 5uL of each sample (~600ng) on a 1.0% agarose, 1x modified TAE gel stained with ethidium bromide.

Gel Layout:

Lane 1 – Hyperladder I (Bioline)

Lane 2 – Claire’s CgF gonad sample

Lane 3 – Mac’s gonad sample

Used Hyperladder I this time, which has a high molecular weight band of 10kb and a low molecular weight band of 200bp.

Well, this totally sucks. Both samples appear to consist of nothing but 150-200bp fragments. Is something actually degrading these samples? The Buffer EB I used during the initial extraction is certainly old. Possible source of degradation? Ugh. Maybe I’ll try this again, but resuspend in TE…

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).

DNA Isolation – Claire’s C.gigas Female Gonad and Mac’s C.gigas Gonad

Due to the poor quality DNA yielded by the DNeasy Kit (Qiagen; see 20140404), I am re-isolating these samples using DNazol (Molecular Research Center). Weighed tissue from each frozen sample:

Claire’s (Female DNA; 5/6/2013) – 0.022g

Mac’s (EV2 9.g) – 0.017g

Incubated samples in 500uL of DNazol + 100ug/mL Proteinase K (2.7uL of 18.5mg/mL stock) O/N at RT on rotator. An additional 500uL of DNazol was added, mixed by pipetting to break up remaining tissues clumps. Manufacturer’s protocol was followed, substituting the first EtOH wash with a wash of 70% DNazol, 30% 100% EtOH. Samples were resuspended in 100uL Buffer EB (Qiagen) and spec’d on a NanoDrop1000.

NOTE: Mac’s sample seemed to get “chunky”/cloudy during the precipitation portion of the procedure. Claire’s remained clear. Although not noted, Mac’s sample behaved in a similar fashion when adding Buffer AL to the sample when using the Qiagen DNeasy Blood & Tissue Kit. Finally, Mac has previously mentioned this behavior to me as well.

Results:

Suprisingly high yields from Mac’s sample.

Both samples exhibit poor 260/230 ratios and high absorbance at 230nm is evident in both samples. Mac’s sample may benefit from

Ran ~600ng of each sample on a 0.8% 1x modified TAE agarose gel to visually assess sample quality.

Gel Loading (from left to right):

  1. Hyperladder II (Bioline)

  2. Claire’s Female DNA

  3. Mac’s gonad (EV2 9.go)

I knew the ladder was of little use due to high molecular weight of gDNA, but it still serves as a bit of a reference. Highest molecular weight band is 2000bp.

Claire’s sample looks pretty good, in relation to the lack of smearing. A single, high molecular weight band is present (albeit, faint) with almost no smearing. However, I’m disappointed by the lack of definition in the band. I fully expected a sharper, more defined band.

Mac’s sample shows a high molecular weight band and significant smearing. Smearing could be indicative of either DNA degradation or high amounts of RNA carryover. If the latter, could explain the high yield.

Will attempt to clean up both samples (RNase and/or do a chloroform clean up).