Tag Archives: DNased RNA

qPCR – Re-run Jake’s O.lurida DNased RNA Samples NC1, SC1, SC2, SC4 from 20150514

The following DNased RNA samples showed inconsistencies between qPCR reps (one rep showed amplification, the other rep did not) on 20150514:

  • NC1
  • SC1
  • SC2
  • SC4

Reran these four samples to obtain a definitive answer as to whether or not they have residual gDNA in them prior to using them to make cDNA.

Used Oly_Actin primers (SR IDs: 1504, 1505)

Used 1μL from all templates.

All samples were run in duplicate.

Positive control was HL1 O.lurida DNA isolated by Jake on 20150323.

Cycling params:

  • 95C – 2.5mins
  • 40 cycles of:
    • 95C – 10s
    • 60C – 20s
  • Melt curve

Master mix calcs: 20150521_qPCR_Oly_DNased_RNA

Plate layout: 20150521_qPCR_plate_Jake_Oly_DNased_RNA

Results:

qPCR Data File (Opticon): Sam_20150521_145749.tad
qPCR Report (Google Sheet): 20150521_qPCR_Report_Jake_Oly_DNased_RNA

 

No amplification in any of the RNA samples, nor the NTCs. Will make cDNA.

 

Amplification Plots

 

 

Melt Curves

qPCR – Jake’s O.lurida ctenidia DNased RNA (1hr Heat Shock Samples)

Ran qPCR on DNased RNA from earlier today to assess whether there was any residual gDNA after the DNase treatment with Oly_Actin_F/R primers (SR IDs: 1505, 1504).

Used 1μL from all templates.

All samples were run in duplicate.

Positive control was HL1 O.lurida DNA isolated by Jake on 20150323.

Cycling params:

  • 95C – 2.5mins
  • 40 cycles of:
    • 95C – 10s
    • 60C – 20s
  • Melt curve

Master mix calcs are here (used same calcs from the other day): 20150512_qPCR_Oly_RNA

Plate layout: 20150514_qPCR_plate_Jake_Oly_1hr_HS_DNased_RNA

Results:

qPCR Data File (Opticon): Sam_20150514_170332.tad

qPCR Report (Google Spreadsheeet): 20150514_qPCR_Report_Jake_Oly_DNased_1hr_HS_RNA

 

Positive control samples are the only samples that produced amplification (cycle ~20). Will proceed to making cDNA.

 

Amplification Plots

 

Melt Curves

qPCR – Jake’s O.lurida ctenidia DNased RNA (Control Samples)

Ran qPCR on DNased RNA from earlier today to assess whether there was any residual gDNA after the DNase treatment with Oly_Actin_F/R primers (SR IDs: 1505, 1504).

Used 1μL from all templates.

All samples were run in duplicate.

Positive control was HL1 O.lurida DNA isolated by Jake on 20150323.

Cycling params:

  • 95C – 2.5mins
  • 40 cycles of:
    • 95C – 10s
    • 60C – 20s
  • Melt curve

Master mix calcs are here: 20150514_qPCR_Oly_DNased_RNA

qPCR Plate Layout: 20150514_qPCR_plate_Jake_Oly_Control_RNA

Results:

qPCR Data File (Opticon): Sam_20150514_153529.tad

qPCR Report (Google Spreadsheet): 20150514_qPCR_Report_Jake_Oly_DNased_Control_RNA

Positive control comes up around cycle ~21.

No amplification in the no template controls.

Four wells of the DNased RNA samples exhibit amplification (B5, C10, C12, D3), however each respective replicate does not. Will re-test these four samples (NC1, SC1, SC2, SC4).

 

Amplification Plots

 

Melt Curves

 

DNase Treatment – Jake’s O.lurida Ctenidia RNA (1hr Heat Shock) from 20150506

Since the O.lurida RNA I isolated on 20150506 showed residual gDNA via qPCR, I treated 1.5μg of RNA from each sample using the Turbo DNA-free Kit (Ambion/Life Technologies), following the “rigorous” protocol.

Briefly:

  • 50μL reactions were carried out in 0.5mL tubes
  • added 1μL of DNase to each tube
  • incubated 30mins @ 37C
  • added additional 1μL of DNased
  • incubated 30mins @ 37C
  • added 0.2 vols (10.2μL) of DNase Inactivation Reagent
  • incubated and mixed for 2mins @ RT
  • transferred 50μL of supe to sterile 1.5mL snap cap tubes
  • spec’d on Roberts Lab NanoDrop1000

Samples were stored @ -80C in Shellfish RNA Box #5 and Box #6.

DNase reaction calcs: 20150514_Jake_Oly_1hr_HS_DNase_calcs

 

 

Results:

Google Spreadsheet: 20150514_DNased_RNA_Jake_Oly_1hr_HS_ODs

 

 

 

 

All samples look pretty good except for HT1 8 (RNA concentration is ridiculously high!) and NT1 8 (RNA concentration is way below expected). Will check for residual gDNA via qPCR.

DNase Treatment – Jake’s O.lurida Ctenidia RNA (Controls) from 20150507

Since the O.lurida RNA I isolated on 20150507 showed residual gDNA via qPCR, I treated 5μg of RNA from each sample using the Turbo DNA-free Kit (Ambion/Life Technologies), following the “rigorous” protocol.

Briefly:

  • 50μL reactions were carried out in 0.5mL tubes
  • added 1μL of DNase to each tube
  • incubated 30mins @ 37C
  • added additional 1μL of DNased
  • incubated 30mins @ 37C
  • added 0.2 vols (10.2μL) of DNase Inactivation Reagent
  • incubated and mixed for 2mins @ RT
  • transferred 50μL of supe to sterile 1.5mL snap cap tubes
  • spec’d on Roberts Lab NanoDrop1000

Samples were stored @ -80C in Shellfish RNA Box #5 and Box #6.

DNase reaction calcs: 20150514_Jake_Oly_control_DNase_calcs

 

 

Results:

 

Google Spreadsheet: 20150514_DNased_RNA_Jake_Oly_controls_ODs

 

 

 

 

Overall, samples look fine. Will check for residual gDNA via qPCR.

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.
  • Shaking incubation step was performed with Disruptor Genie
  • 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.

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.
  • Shaking incubation step was performed with Disruptor Genie
  • 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.

qPCR – Check DNased RNA from Earlier Today for Residual gDNA

Ran qPCR using V.tubiashii VtpA primers (from Elene; no SR ID). Used 0.5uL of each DNased RNA sample, which equals ~40ng of RNA, which would be the equivalent amount of RNA that would end up in a qPCR rxn after cDNA has been made (using 1uL of cDNA). Used the filter DNA extraction from samples #279 from DATE as a positive control. Master mix calcs are here. Plate layout, cycling params, etc. can be found in the qPCR Report (see Results).

Results:

qPCR Data File (CFX96)

qPCR Report (PDF)

All samples showed up negative, except for the positive control. Will proceed with making cDNA on Monday.

Reverse Transcription – Dave’s Manila Clam (Venerupis philippinarum) DNased RNA from 20120307 and 20120302

Performed reverse transcription on 1.5ug of DNased RNA in a 75uL reaction, using oligo dT primers. All reagents were scaled appropriately (based on Promega’s M-MLV RT protocol). Samples were prepared in a plate and stored @ -20C. Plate layout and all reverse transcription calcs are here:

qPCR – Dave’s Manila Calm (Venerupis philippinarum) DNased RNA from yesterday and 20120302

Performed qPCR on all DNased RNA samples from this group (samples #1-48) using beta actin primers (SR IDs: 1379, 1380). 0.5uL of each DNased RNA was used, which was the equivalent of ~40ng, in order to simulate the amount of RNA present in the subsequent cDNA (1000ng of RNA in 25uL cDNA; use 1uL of cDNA in qPCR reaction). Master mix calcs are here. Plate layout, cycling params, etc., can be found in the qPCR Report (see Results). 0.5uL of total RNA from sample Vp gill 01 was used to serve as a positive control, since Dave has no existing V. phillippinarum cDNA.

Results:

qPCR Data File (CFX96)

qPCR Report (PDF)

All samples are clean and are ready for reverse transcription.

Of note, the overall fluorescence of the reactions was very low. As such, the default baseline analysis setting (linear) suggested that all samples had a Cq value because the baseline was incorrectly set an was NOT above background fluorescence levels. Changing the baseline analysis setting to “regression” resolved this. Also, it should be noted that one sample (#48) other than the positive control actually does show amplification and a corresponding melt curve. However, the melt curve peak is at a different temp than the positive control, suggesting that this is non-specific amplification in sample #48.