Tag Archives: mechanical stress

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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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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