Tag Archives: RNA isolation

RNA Isolation – Abalone Water Filters for RLO Viability

Water filters stored at -80C in ~1mL of RNAzol RT were provided by Lisa. This is part of an experiment (and Capstone project) to assess RLO viability outside of the host.

The samples were thawed and briefly homogenized (as best I could) with a disposable plastic pestle. The samples were then processed according to the manufacturer’s protocol for total RNA isolation. Samples were resuspended in 25μL of nuclease-free water (Promega).

Immediately proceeded to DNase treatment.

The experimental samples and the various treatments are viewable in the “Viability Trial 3″ tab of Lisa’s spreadsheet (Google Sheet): RLO Viability & ID50

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RNA Isolation – Abalone Water Filters

Lisa provided me with a set of water filters stored in 1mL RNAzol RT @ -80C to isolate RNA from. This is an attempt to assess withering syndrome viability from within the water.

The samples were thawed and briefly homogenized (as best I could) with a disposable plastic pestle. The samples were then processed according to the manufacturer’s protocol for total RNA isolation. Samples were resuspended in 20μL of 0.1%-DEPC H2O.

Samples were stored @ -80C. Will DNase next week.

The sample names are as follows (the ‘C’ is short for “Control”, per Lisa):

  • T0A
  • T0B
  • T1A
  • T1A C
  • T1B C
  • T3A
  • T3B
  • T7A
  • T7B
  • T7A C
  • T7B C

 

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RNA Isolation – O.lurida Ctenidia 1hr Post-Mechanical Stress

Isolated RNA from Jake’s Ostrea lurida ctenidia samples that had been subjected to mechanical stress (from 20150422).

Despite the indication in this notebook, the samples had not been previously homogenized in RNAzol RT. I thawed the samples, homogenized them and followed the RNAzol RT protocol for total RNA isolation. Here’s the list of samples:

  • 42215 HM1 1
  • 42215 HM1 2
  • 42215 HM1 3
  • 42215 HM1 4
  • 42215 HM1 5
  • 42215 HM1 6
  • 42215 HM1 7
  • 42215 HM1 8
  • 42215 SM1 1
  • 42215 SM1 2
  • 42215 SM1 3
  • 42215 SM1 4
  • 42215 SM1 5
  • 42215 SM1 6
  • 42215 SM1 7
  • 42215 SM1 8

RNA was resuspended in 50μL of 0.1%DEPC-H2O and stored @ -80C in the original box they came from.

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RNA Isolation – O.lurida Ctenidia 1hr Post-Mechanical Stress

Isolated RNA from Jake’s Ostrea lurida ctenidia samples that had been subjected to mechanical stress (from 20150422).

Despite the indication in this notebook, the samples had not been previously homogenized in RNAzol RT. I thawed the samples, homogenized them and followed the RNAzol RT protocol for total RNA isolation. Here’s the list of samples:

  • 42215 NM1 1
  • 42215 NM1 2
  • 42215 NM1 3
  • 42215 NM1 4
  • 42215 NM1 5
  • 42215 NM1 6
  • 42215 NM1 7
  • 42215 NM1 8

RNA was resuspended in 50μL of 0.1%DEPC-H2O and stored @ -80C in the original box they came from.

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RNA Isolation – Geoduck Gonad in Paraffin Histology Blocks

UPDATE 20150528: The RNA isolated in this notebook entry may have been consolidated on 20150528.

The RNA isolation I performed earlier this week proved to be better for some of the samples (scraping tissue directly from the blocks), but still exhibited low yields from some samples. I will perform a final RNA isolation attempt (the kit only has six columns left) from the following samples:

  • 02
  • 03
  • 04
  • 07
  • 08
  • 09

Instead of full sections from each histology cassette, I gouged samples directly from the tissue in each of the blocks to maximize the amount of tissue input.

IMPORTANT:

Samples were then processed with the PAXgene Tissue RNA Kit in a single group.

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 40μL of Buffer TR4, incubated @ 65C for 5mins, immediately placed on ice and quantified on the Roberts Lab NanoDrop1000.

 

All samples were stored @ -80C in Shellfish RNA Box #5.

Results:

 

Two samples (02 and 07) produced great yields and perfect RNA (260/280 and 260/230 of ~2.0). The remainder of the samples showed little improvement compared to what I’ve been obtaining from the previous three attempts. Will discuss with Steven and Brent about how to proceed with this project.

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RNA Isolation – Jake’s O. lurida Ctenidia Control from 20150422

Isolated RNA from Jake’s Olympia oyster ctenidia, controls, collected on 20150422. Samples had been homogenized and stored @ -80C.

The following sample tubes (heat-shocked oyster ctenidia) were removed from -80C and thawed at RT:

  •  42215 HC 1
  •  42215 HC 2
  • 42215 HC 3
  • 42215 HC 4
  • 42215 HC 5
  • 42215 HC 6
  • 42215 HC 7
  • 42215 HC 8
  • 42215 NC 1
  • 42215 NC 2
  • 42215 NC 3
  • 42215 NC 4
  • 42215 NC 5
  • 42215 NC 6
  • 42215 NC 7
  • 42215 NC 8
  • 42215 SC 1
  • 42215 SC 2
  • 42215 SC 3
  • 42215 SC 4
  • 42215 SC 5
  • 42215 SC 6
  • 42215 SC 7
  • 42215 SC 8

 

NOTE: 0.1% DEPC-H2O used throughout this procedure was prepared on 7/15/2010 by me.

 

According to Jake’s notebook entry, the samples should have been previously homogenized in RNAzol RT (Molecular Research Center; MRC). However, none of the samples showed evidence of being homogenized:

 

 

 

Procedure:

Samples were homogenized with disposable pestle in their respective tubes and vortexed.

Added 400μL of 0.1% DEPC-H2O to each sample and vortexed 15s.

Incubated samples 15mins at RT.

Centrifuged tubes 15mins at RT @ 16,000g.

750μL of the supe was transferred to a clean tube, added equal volume of isopropanol (750μL), mixed by inversion (20 times), and incubated at RT for 15mins.

Centrifuged 12,000g for 10mins.

Discarded supe.

Washed pellets with 500μL of 75% EtOH (made with 0.1% DEPC-H2O) and centrifuged 4,000g for 3mins at RT. Repeated one time.

Removed EtOH and resuspended in 100μL of 0.1% DEPC-H2O. Most samples required vortexing to dissolve pellet.

Sample tubes were transferred to ice, quantified on the Roberts Lab NanoDrop1000, and stored @ -80C in their original box, pictured:

 

 

 

 

Results:

Google Spreadsheet with absorbance data: 20150507_Jake_Oly_control_RNA_ODs

 

Excellent yields and pretty solid 260/280 ratios (>1.85). Interestingly, the 260/230 ratios aren’t so great (compared to yesterday’s isolations). I suspect that the reason for this is that there appeared to be more starting tissue in these samples than yesterday’s. The greater quantity of tissue explains the higher yields and could be tied to the decrease in the 260/230 ratios…

Anyway, things look good. Next step will be to check for gDNA carryover in these samples and yesterday’s samples.

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RNA Isolation – Jake’s O. lurida Ctenidia 1hr Heat Stress from 20150422

Isolated RNA from Jake’s Olympia oyster ctenidia, 1hr heat shock, collected on 20150422. Samples had been homogenized and stored @ -80C.

The following sample tubes (heat-shocked oyster ctenidia) were removed from -80C and thawed at RT:

  • 42215 HT1 1
  • 42215 HT1 2
  • 42215 HT1 3
  • 42215 HT1 4
  • 42215 HT1 5
  • 42215 HT1 6
  • 42215 HT1 7
  • 42215 HT1 8
  • 42215 NT1 1
  • 42215 NT1 1
  • 42215 NT1 2
  • 42215 NT1 3
  • 42215 NT1 4
  • 42215 NT1 5
  • 42215 NT1 6
  • 42215 NT1 7
  • 42215 NT1 8
  • 42215 ST1 1
  • 42215 ST1 2
  • 42215 ST1 3
  • 42215 ST1 4
  • 42215 ST1 5
  • 42215 ST1 6
  • 42215 ST1 7
  • 42215 ST1 8

NOTE: Samples NT1 1 and NT1 2 only had 700μL of RNAzol RT in them. Added additional 300μL of RNAzol RT to each.

NOTE: 0.1% DEPC-H2O used throughout this procedure was prepared on 7/15/2010 by me.

According to Jake’s notebook entry, the samples should have been previously homogenized in RNAzol RT. However, none of the samples showed evidence of being homogenized:

 

In theory, if these samples were snap frozen on liquid nitrogen after being placed in the RNAzol RT, there should be almost no impact on the RNA.

 

Procedure:

Samples were homogenized with disposable pestle in their respective tubes and vortexed.

Added 400μL of 0.1% DEPC-H2O to each sample and vortexed 15s.

Incubated samples 15mins at RT.

Centrifuged tubes 15mins at RT @ 16,000g.

750μL of the supe was transferred to a clean tube, added equal volume of isopropanol (750μL), mix by inversion (20 times), and incubated at RT for 15mins.

Centrifuged 12,000g for 10mins.

Discarded supe.

Washed pellets with 500μL of 75% EtOH (made with 0.1% DEPC-H2O) and centrifuged 4,000g for 3mins at RT. Repeated one time.

Removed EtOH and resuspended in  100μL of 0.1% DEPC-H2O. Most samples required vortexing to dissolve pellet.

Sample tubes were transferred to ice, quantified on the Roberts Lab NanoDrop1000, and stored @ -80C in their original box, pictured:

 

Results:

 

Google Spreadsheet with absorbance data: 20150506_Jake_Oly_1h_HS_RNA_ODs

Overall, the samples have excellent yields. The exceptions being the two samples that had less than 1mL of RNAzol RT in them to start (their yields are actually fine, but relative to all the other samples, they aren’t great). Should I have left them that way instead of adding additional RNAzol RT? Was there something wrong with these samples in the first place and that’s why they didn’t have a full 1mL of RNAzol RT in the tube already?

The 260/280 ratios are pretty good for most of the samples (>1.8), however I’d prefer to see RNA with 260/280 ratios >1.9.

The 260/230 ratios are amazing! The best I’ve seen coming straight out of an RNA isolation in a long time.

Eventually (once I’ve isolated RNA from the control set that corresponds to these heat shock samples), I’ll check for gDNA carryover and then, probably, DNase the RNA.

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RNA Isolation – Geoduck Gonad in Paraffin Histology Blocks

UPDATE 20150528: The RNA isolated in this notebook entry may have been consolidated on 20150528.

Last week’s RNA isolation (a second attempt at obtaining RNA from the samples) performed poorly. I will re-isolate RNA from the following samples:

  • 02
  • 03
  • 04
  • 07
  • 08
  • 09
  • 35
  • 38
  • 46
  • 65
  • 67
  • 68

Instead of full sections from each histology cassette, I gouged/shaved off samples directly from the tissue in each of the blocks to maximize the amount of tissue input. However, due to the small size and susceptibility to flying around because of static electricity, none of these were able to be weighed prior to processing.

IMPORTANT:

Samples were then processed with the PAXgene Tissue RNA Kit in a single group.

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 40μL of Buffer TR4, incubated @ 65C for 5mins, immediately placed on ice and quantified on the Roberts Lab NanoDrop1000.

Results:

 

 

 

Well, despite the low numbers, all of the samples (excluding 46 – 68) are double the yield of what I saw previously. This is good, but the amount of RNA from these is probably borderline sufficient quantity for RNA-Seq.

The kit has enough columns for six sample preps. I think I’ll attempt this strategy again (gouging/shaving directly from tissue in histo cassette), but really take a fair amount of tissue this time and see if I can get more.

All samples were stored @ -80C in Shellfish RNA Box #5.

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RNA Isolation – Geoduck Gonad in Paraffin Histology Blocks

UPDATE 20150528: The RNA isolated in this notebook entry may have been consolidated on 20150528.

Last week’s RNA isolation failed for more than half of the samples I processed. I will re-isolate RNA from the following samples:

  • 02
  • 03
  • 04
  • 07
  • 08
  • 09
  • 35
  • 38
  • 46
  • 65
  • 67
  • 68

IMPORTANT:

Five 5μm sections were taken from each block. A new blade was used for each block.

Samples were then processed with the PAXgene Tissue RNA Kit in two groups of six.

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 40μL of Buffer TR4, incubated @ 65C for 5mins, immediately placed on ice and quantified on the Roberts Lab NanoDrop1000.

Results:

 

 

 

Well, these results are very consistent with the data from the last isolation performed on these samples. This fact suggests that the problem lies with the tissue samples and not the isolation (since the isolation has been performed two separate times on these same samples and the results have come out virtually identical both times).

All samples with concentrations < 5ng/μL were discarded. The remaining samples were stored @ -80C in Shellfish RNA Box #5:

  • 35
  • 38
  • 65
  • 67

Will discuss with Steven, look at Grace’s notebook to review the preservation process for these samples, and review the PAXgene Tissue RNA Kit to see if it will accommodate a greater number of microtome sections to use for isolation.

 

 

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RNA Isolation – Geoduck Gonad in Paraffin Histology Blocks

UPDATE 20150528: The RNA isolated in this notebook entry may have been consolidated on 20150528.

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 the following samples using the PAXgene Tissue RNA Kit (Qiagen) from the following geoduck sample blocks:

  • 02
  • 03
  • 04
  • 07
  • 08
  • 09
  • 35
  • 38
  • 41
  • 46
  • 51
  • 65
  • 67
  • 68
  • 69
  • 70

IMPORTANT:

Five 5μm sections were taken from each block. A new blade was used for each block.

Samples were then processed with the PAXgene Tissue RNA Kit in two groups of eight.

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 40μL of Buffer TR4, incubated @ 65C for 5mins, immediately placed on ice and quantified on the Roberts Lab NanoDrop1000.

Results:

 

 

Well, these results are certainly not good.

The first set of eight samples I processed yielded no RNA (except #38, which is only marginally better than nothing). All the samples (excluding #38) have been discarded.

The second set of eight samples I processed range from amazing to poor (#68 was barely worth keeping).

I’ll review the protocol, but at the moment I’m at a loss to explain why the first set of eight samples came up empty. Will perform another on these blocks on Monday. Grrrrr.

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

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