Tag Archives: ctenidia

Reverse Transcription – Subset of Jake’s O.lurida DNased RNA

Currently don’t have sufficient reagents to perform reverse transcription on the entire set of DNased RNA (control and 1hr.heat-shocked O.lurida ctenidia samples). To enable Jake to start testing out some of his primers while we wait for reagents to come in, Steven suggested I generate some cDNA for him to use.

Used the following DNased RNA:

  • HC1
  • NC1
  • SC1
  • HT1 1
  • NT1 1
  • ST1 1

Reverse Transcription Calcs: 20150522_Jake_Oly_cDNA_Calcs

Briefly:

  • Reactions run in 0.5mL snap cap tubes
  • 250ng of DNased RNA used in each reaction
  • Combined DNased RNA with oligo dT primers and water; incubated 70C 5mins; immediately placed on ice
  • Added 6.75μL of buffer/dNTP/enzyme master mix to each sample; incubated 42C for 1hr; 95C for 3mins

Samples will be given to Jake and stored @ -20C.

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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

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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

 

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qPCR – Jake O.lurida ctenidia RNA (Heat Shock Samples) from 20150506

Ran qPCRs on the O.lurida total RNA I isolated on 20150506 to assess presence of gDNA carryover with Oly Actin 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.

Master mix calcs are here: 20150512_qPCR_Oly_RNA

Cycling params:

  • 95C – 3mins
  • 40 cycles of:
    • 95C – 5s
    • 60C – 20s
  • Melt curve

 

Plate layout: 20150512_qPCR_plate_Jake_Oly_HS_RNA

Results:

qPCR Data File (Opticon2): Sam_20150512_123246.tad

qPCR Report (Google Spreadsheet):20150512_qPCR_Report_Jake_Oly_HS_RNA

Excluding the no template controls (NTC), all samples produced amplification. Will require DNasing before making cDNA.

Related to the qPCR I ran earlier today with these same primers, the efficiencies of the reactions on this plate are significantly better (i.e. normal; >80% efficiencies) than the earlier qPCR. The improved efficiency would also explain why the positive control comes up two cycles earlier on this run.

In the amplification plots below, the positive control reps are the two lines coming up at cycle ~20.

 

Amplification Plots

 

Melt Curves

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qPCR – Jake O.lurida ctenidia RNA (Control Samples) From 20150507

Ran qPCRs on the O.lurida total RNA I isolated on 20150507 to assess presence of gDNA carryover with Oly Actin 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.

Master mix calcs are here: 20150512_qPCR_Oly_RNA

Cycling params:

  • 95C – 3mins

40 cycles of:

  • 95C – 5s
  • 60C – 20s

Melt curve

 

Plate layout: 20150512_qPCR_plate_Jake_Oly_Control_RNA

 

Results:

qPCR Data File (Opticon2): Sam_20150512_105811.tad

qPCR Report (Google Spreadsheet): 20150512_qPCR_Report_Jake_Oly_Control_RNA

Excluding the no template controls (NTC), all samples produced amplification. Will require DNasing before making cDNA.

On a side note, it should be noted that the efficiencies for all of the reactions were pretty bad; probably averaging 50%. Not entirely sure why or what that indicates.

In the amplification plots below, the positive control reps are the two red lines coming up at cycle ~22.

Amplification Plots

 

 

Melt Curves

 

 

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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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