Tag Archives: gill

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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RNA Isolation – Dave’s Manila Clam (Venerupis philippinarum) Gill Samples (#1-24)

Isolated RNA from Manila Clam gill samples provided by Dave according to protocol. Samples were resuspended in 0.1%-DEPC H2O and spec’d on the Roberts Lab NanoDrop1000. Samples were stored @ -80C in Dave’s box that the tissue was initially stored in.

Results:

Overall, RNA quality is very good, as well as yields.

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RNA Isolation – Hard Clam Gill Tissue from Vibrio Experiment (see Dave’s Notebook 5/2/2011)

Isolated RNA in 1mL of Tri-Reagent according to manufacturer’s protocol. Samples were precipitated with isopropanol and stored over the weekend @ -20C. Will conclude isolation on Monday. The samples isolated were:

MA 1-11

MA Vt 1-11

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qPCR – C.gigas COX1/COX2 Tissue Distribution

Performed qPCR using pooled cDNA from 20110311. Pooled 2uL from each of the following samples groups: Dg 3hr C, Gill 1hr C, Gill 1hr E, Mantle 3hr C, and Muscle 3hr C. Master mix calcs are here. Plate layout, cycling params, etc can be found in the qPCR Report (see Results). Primers sets run were:

EF1_qPCR_5′,3′ (SR IDs: 309, 310)

Cg_COX1/2_qPCR_F (SR ID: 1192) + Cg_COX1_qPCR_R (SR ID: 1191)- Target = COX1

Cg_COX1/2_qPCR_F (SR ID: 1192) + Cg_COX2_454align1_R (SR ID: 1190) – Target = COX2

Results:

qPCR Report (PDF)

qPCR Data File (CFX96)

Graphs were generated using the BioRad CFX Manager v2.0 software. Expression was normalized to EF1. Also to note, gene efficiency was assumed as 100% by the software since no standard curve was run on the plate. As such, analysis of this data may not be exact.

It’s clear by examining the graphs that the primers being used to differentiate COX1 and COX2 (since they share a common primer: SRID 1192) are differentially expressed. This indicates that the primer sets are indeed amplifying different targets as hoped. This was the primary intention of this qPCR. However, we also now have an idea of tissue distribution of the two genes, as well as their response to V. vulnificus exposre after 1hr. Next step is to perform this qPCR on all the individuals from this experiment as well as the different tissues.

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DNase – DNase C.gigas RNA from 20110120, 20110121 and 20110124

5ug of RNA was DNased using Ambion’s Turbo DNA-free kit, following the rigorous protocol (0.5uL of DNase for 30 mins then additional 0.5uL of DNase for 30mins). Calcs for DNase reactions are here. RNA was stored @ -80C in Shellfish RNA Boxes 4 and 5. Samples will be spec’d on Monday.

Results:

Overall, the RNA looks really good (based on OD 260/280 numbers). Not surprisingly, the OD 260/230 values for all samples dropped, likely due to the addition of the buffer (salts) used in the DNase reaction. Emma says she will check these samples for residual DNA.

–UPDATE (20110131)– Emma checked all DNased RNA samples on 20110131 using C.gigas 18s primers (SR ID: ?). She has not listed the results of the whether or not all samples are clean or if some still have residual gDNA carryover.

–UDPATE (20110201– Samples that appear to have residual gDNA carryover based on Emma’s qPCR on 20110131: Muscle C6, Gill 1hr C2 & E2.

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RNA Isolation – Various C.gigas Tissue from 20110111

RNA was isolated in 1mL TriReagent, according to protocol. Samples were resuspended in 50uL 0.1% DEPC-H2O and spec’d. RNA was stored @ -80C in “Shellfish RNA Box #4“.

Results:

Overall, all RNA looks very good (based on 260/280 and 260/230 values).

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