Tag Archives: ctenidia

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

Quantified the RNA I isolated from Jake’s samples on 20150715 and 20150710 using the Roberts Lab NanoDrop1000 (ThermoFisher).

 

 

 

Overall, the yields are good. The 260/280 ratios are mediocre. Will proceed with DNase treatment.

DNased 1.5ug 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
  • spun 1.5mins, 10,000g @ 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 #6. Will quantify at a later date.

DNase reaction calcs: 20150727_Jake_Oly_mech_stress_DNase_calcs

 

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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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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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