Tag Archives: DNased RNA

Samples Received – Pinto Abalone DNased RNA from UC-Irvine

Received DNased pinto abalone RNA from Alyssa Braciszewski at UC-Irvine. These are subset of the samples I sent her back in February.

Here’s the samples list provided by Alyssa (Google Sheet): shipment to UW of RNA samples.xlsx

The samples need to be confirmed to be free if residual RLO gDNA via qPCR. If they are clean, then will proceed to making cDNA, using provided reagents.

Reagents were stored in door of -20C in FSH 240.

Samples were stored in the provided box in the “new” -80C in FSH 235.

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DNA Quantification – RLO viability DNased RNA

I previously DNased RNA I isolated from water filters that were part of the RLO viability experiment that Lisa and the Capstone students are conducting. I checked for residual gDNA carryover via qPCR and all of the samples that were intended for dosing the abalone came up positive. It’s likely due to such a high quantity of algae that was co-filtered with the potential RLOs, leading to over-saturation of the RNAzol with DNA, resulting in the gDNA carryover.

In turn, I think the DNase treatment was insufficient for the quantity of carryover DNA.

I am planning on re-DNasing those samples, but want to quantify any residual DNA present to make sure that the samples aren’t still too concentrated for the DNase.

Samples were quantified using the Robert Lab Qubit 3.0 and the Qubit dsHS reagents (high sensitivity), using 1uL of sample.

Results:

Residual DNA is still present, but at levels that are well below the maximum that the DNase treatment (10ug) can handle. I will redo the DNase treatment on these samples. Spreadsheet is linked, and embedded below, with sample concentrations.

Spreadsheet (Google Sheet): 20170424_filter_rna_dna_quant

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qPCR – Capstone RLO Viability DNased RNA

Need to verify that the DNased RNA I made previously does not have any detectable gDNA present.

Ran the withering syndrome qPCR assay on the DNased RNA.

Standard curve was p18RK7 from 20161128.

All samples were run in duplicate. As such, the number of samples required to qPCR runs.

Master mix calcs are here (Google Sheet): 20170406_qPCR_WSN1_capstone

Plate layout, cycling params, etc. can be found in the qPCR Report (see Results).

Baseline threshold was manually set to 580, as previously determined by Lisa.

Results:

qPCR Report (PDF): Sam_2017-04-06 10-01-23_CC009827.pdf
qPCR Data File (CFX96): Sam_2017-04-06 10-01-23_CC009827.pcrd

qPCR Report (PDF): Sam_2017-04-06 11-36-53_CC009827_capstone_RLO_viability_WSN1.pdf
qPCR Data File (CFX96): Sam_2017-04-06 11-36-53_CC009827_capstone_RLO_viability_WSN1.pcrd

Well, some samples came up positive for residual DNA. The samples that came up positive are all three dilutions of the RLO used for initial infection of the abalone.

This makes things interesting to deal with. Seeing that no other samples have detectable DNA suggests that those samples are fine to move forward with for reverse transcription. However, it’s unlikely that the DNase treatment only worked on a subset of a samples, since it was distributed via a master mix.

Regardless, there isn’t any additional RNA to work with. So, I’ll put the samples that came up positive through a second round of DNase treatment. Addtionally, I may dilute them slightly to avoid complications from accumulation of too much DNase buffer, due to leftover buffer from the first round…


Amplification Plots from Sam_2017-04-06 10-01-23_CC009827.pcrd

Green = p18RK7 standards
Blue = samples
Red = No template control

 

Standard Curve from Sam_2017-04-06 10-01-23_CC009827.pcrd

 

 

Amplification Plots from Sam_2017-04-06 11-36-53_CC009827_capstone_RLO_viability_WSN1.pcrd

 

 

Standard Curve from Sam_2017-04-06 11-36-53_CC009827_capstone_RLO_viability_WSN1.pcrd

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DNase Treatment – Abalone Water Filters for RLO Viability

The RNA I isolated earlier today was subjected to DNase treatment using the Turbo DNA-free Kit (Invitrogen), following the manufacturer’s standard protocol.

After DNase inactivation treatment, the RNA was transferred (recovered ~19uL from each samples)  to a clear, low-profile PCR plate.

The plate layout is here (Google Sheet): 20170309_RLO_viability_DNased_RNA_plate_layout

The samples will be subjected to qPCR to assess the presence/absence of residual gDNA. The plate of DNased RNA was stored @ -80C in the original box that the water filters were stored in.

An overview of the experiment and the various treatments are viewable in the “Viability Trial 2″ tab of Lisa’s spreadsheet (Google Sheet): RLO Viability & ID50

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Reverse Transcription – Water Filter DNased RNA (from 20161207)

Performed reverse transcription on the DNased RNA samples that I verified were free of detectable RLO DNA (20161207).

Combined 17μL DNased RNA + 0.5μL random primers (Promega; Cat: C1181) in 0.2mL PCR tubes.

NOTE: The 17μL was virtually all of the sample volume recovered from DNasing. As such, the DNased RNA will not be quantified.

Incubated DNased RNA and primer mix in PTC-200 thermal cycler (MJ Research) at 70C for 5mins w/heated lid, then immediately placed on ice.

Created master mix of following components:

REAGENT SINGLE REACTION VOL (μL) NUMBER REACTIONS TOTAL VOL (μL)
5x MMLV RT BUFFER 5 14 70
10mM dNTPS (Promega) 1.25 14 17.5
MMLV RT (Promega) 0.5 14 7

Added 6.75 of master mix to each and mixed by pipetting.

Incubated PTC-200 thermal cycler (MJ Research) @ 37C for 1hr (no heated lid), followed by 95C for 3mins (heated lid). Samples were transferred to 0.5mL snap cap tubes and labelled with “cDNA” and the corresponding sample name. Samples will be stored in my -20C box.

UPDATE 20170830 Lisa has moved these samples to a -20C box dedicated to RLO Viability cDNA.

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qPCR – DNased RNA from Abalone Water Filters (from earlier today)

Prior to creating cDNA, need to verify that the DNased RNA from earlier today doesn’t contain any detectable RLO DNA.

Master mix calcs (Google Sheet): 20161128 – qPCR Water Filter DNased RNA

All samples were run in duplicate. Plate layout, cycling params, etc. are in the qPCR Report (see Results below).

Standard curve was the p18RK7 curve made on 20161128.

Baseline threshold was manually set to 580, as previously determined by Lisa for this assay.

Results:
qPCR Report (PDF): Sam_2016-12-07 09-10-07_CC009827.pdf
qPCR File (CFX96):Sam_2016-12-07 09-10-07_CC009827.pcrd

Standard curve looks good.

No samples amplified. This suggests that there is no detectable DNA in any of the DNased RNA samples. Will proceed with making cDNA.

 

 

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Reverse Transcription – O.lurida DNased RNA 1hr post-mechanical stress

Performed reverse transcription on the Olympia oyster DNased RNA from the 1hr post-mechanical stress samples from Jake’s project. To accommodate the large numbers of anticipated genes to be targeted in subsequent qPCRs, I prepared 100μL reactions (normally, 25μL reactions are prepared) using 250ng of each DNased RNA. A 1:10 dilution of the oligo dT primers (Promega) was prepared to improve pipetting accuracy. All incubations were performed in a thermal cycler without using a heated lid.

DNased RNA was combined with NanoPure H2O and oligo dT primers in 24 wells of a PCR plate, heated @ 70C for 10mins and immediately placed on ice. After 5mins, the plate was spun 2000g @ RT for 2mins and returned to ice.

25.25μL of a master mix containing 5x M-MLV Buffer (Promega), dNTPs (10mM each; Promega), and M-MLV Reverse Transcriptase (50U/rxn; Promega) was distributed to each well and mixed via pipetting. The plate was heated @ 42C for 1hr, 95C for 3mins. The plate was spun 2000g @ RT for 2mins and then stored @ -20C.

Plate layout and all calculations can be found here (Google Sheet): 20150806_Jake_oly_mech_stress_cDNA_calcs

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qPCR – Jake’s O.lurida ctenidia 1hr post-mechanical stress DNased RNA

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: 201500806_qPCR_Oly_DNased_RNA

qPCR Plate Layout: 20150806_qPCR_plate_Jake_Oly_DNased_RNA

Results:

qPCR Data File (Opticon): 20150806_165044.tad
qPCR Report (Google Spreadsheet): 20150806_qPCR_Report_Jake_Oly_DNased_RNA

Positive control comes up around cycle ~21.

No amplification in the no template controls.

Two wells of the DNased RNA samples exhibit amplification (E3, F6), however the corresponding respective replicate does not. Will proceed with reverse transcription.

 

Amplification Plots

Positive Controls

 

Melt Curves

Positive Controls (HL1)

DNased RNA Samples

Follow the green and red lines with the vertical bars. The different colors reflect that those are two different samples. Additionally, their respective replicates do not exhibit amplification.

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RNA Quantification – O.lurida 1hr post-mechanical heat stress DNased RNA

DNased RNA from 07272015 was quantified using the Roberts Lab NanoDrop1000.

 

Results:

The 260/280 ratios don’t look great, but that is most likely due to the DNase treatment. The DNase that’s added to each sample isn’t actually removed, so that additional protein will skew the 260/280 ratios. Will proceed with qPCR to check for any residual gDNA in these samples.

 

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Reverse Transcription – O.lurida DNased RNA Controls and 1hr Heat Shock

Performed reverse transcription on the Olympia oyster DNased RNA from the control samples and the 1hr heat shock samples of Jake’s project. To accommodate the large numbers of anticipated genes to be targeted in subsequent qPCRs, I prepared 100μL reactions (normally, 25μL reactions are prepared) using 250ng of each DNased RNA. A 1:10 dilution of the oligo dT primers (Promega) was prepared to improve pipetting accuracy. All incubations were performed in a thermal cycler without using a heated lid.

DNased RNA was combined with NanoPure H2O and oligo dT primers in 48 wells of a PCR plate, heated @ 70C for 10mins and immediately placed on ice. After 5mins, the plate was spun 2000g @ RT for 2mins and returned to ice.

25.25μL of a master mix containing 5x M-MLV Buffer (Promega), dNTPs (10mM each; Promega), and M-MLV Reverse Transcriptase (50U/rxn; Promega) was distributed to each well and mixed via pipetting. The plate was heated @ 42C for 1hr, 95C for 3mins. The plate was spun 2000g @ RT for 2mins and then stored @ -20C.

Plate layout and all calculations can be found here (Google Sheet): 20150616_Jake_Oly_cDNA_Calcs

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