Tag Archives: RNAzol RT

RNA Isolation & Quantification – Tanner crab hemolymph

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)

Sample ID Conc. (ng/uL) Total Yield (ng)
3859/3656 0.44 22
3665/3873 1.66 83
3665/3873 2.04 102

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

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

Share

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 2″ tab of Lisa’s spreadsheet (Google Sheet): RLO Viability & ID50

Share

Sample Prep – Pinto Abalone Tissue/RNA for Collabs at UC-Irvine

We need to send half of each sample that we have from Sean Bennett’s Capstone project to Alyssa Braciszewski at UC-Irvine.

This is quite the project! There are ~75 samples, and about half of those are tissues (presumably digestive gland) stored in RNAzol RT. The remainder are RNA that has already been isolated. Additionally, tube labels are not always clear and there are duplicates. All of these factors led to this taking an entire day in order to decipher and process all the samples.

I selected samples from only those that I was confident in their identity.

I aliquoted 25μL of each RNA for shipment to Alyssa.

Tissue samples were thawed and tissue was cut in half using razor blades.

Planning to send samples on Monday.

Lisa has already assembled a master spreadsheet to try to keep track of all the samples and what they are (Google Sheet): Pinto Transcriptome

Here’s the list of samples I’ll be sending to Alyssa (Google Sheet): 20170222_pinto_abalone_samples

Here are some images to detail some of the issues I had to deal with in sample ID/selection.

 

 

 

 

 

 

 

 

 

Share

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

 

Share

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.

Share

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.

Share

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.

Share

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.

Share