Tag Archives: Pacific oyster

qPCRs – Ronit’s C.gigas ploidy/dessication/heat stress cDNA (1:5 dilution)

IMPORTANT: The cDNA used for the qPCRs described below was a 1:5 dilution of Ronit’s cDNA made 20181017 with the following primers! Diluted cDNA was stored in his -20oC box with his original cDNA.

The following primers were used:

18s

  • Cg_18s_F (SR ID: 1408)

  • Cg_18s_R (SR ID: 1409)

EF1 (elongation factor 1)

  • EF1_qPCR_5′ (SR ID: 309)

  • EF1_qPCR_3′ (SR ID: 308)

HSC70 (heat shock cognate 70)

  • Cg_hsc70_F (SR ID: 1396)

  • Cg_hsc70_R2 (SR ID: 1416)

HSP90 (heat shock protein 90)

  • Cg_Hsp90_F (SR ID: 1532)

  • Cg_Hsp90_R (SR ID: 1533)

DNMT1 (DNA methyltransferase 1)

  • Cg_DNMT1_F (SR ID: 1511)

  • Cg_DNMT1_R (SR ID: 1510)

Prx6 (peroxiredoxin 6)

  • Cg_Prx6_F (SR ID: 1381)

  • Cg_Prx6_R (SR ID: 1382)

Samples were run on Roberts Lab CFX Connect (BioRad). All samples were run in duplicate. See qPCR Report (Results section) for plate layout, cycling params, etc.

qPCR master mix calcs (Google Sheet):


RESULTS

No analysis here. Will analyze data and post in different notebook entry. This entry just contains the qPCR setup, resulting data, and a glimpse of how each primer performed.

Nothing is broken down based on sample ploidy or experimental conditions.

18s

qPCR Report (PDF):

qPCR File (PCRD):

qPCR Data (CSV):

Amplication Plots

Melt Curves


DNMT1

qPCR Report (PDF):

qPCR File (PCRD):

qPCR Data (CSV):

Amplication Plots

Melt Curves


EF1

qPCR Report (PDF):

qPCR File (PCRD):

qPCR Data (CSV):

Amplication Plots – Manual Threshold (Linear)

Amplication Plots – Manual Threshold (Log)

Amplication Plots – Automatic Threshold (Linear)

Amplication Plots – Automatic Threshold (Log)

Melt Curves


HSC70

qPCR Report (PDF):

qPCR File (PCRD):

qPCR Data (CSV):

Amplication Plots

Melt Curves


HSP90

qPCR Report (PDF):

qPCR File (PCRD):

qPCR Data (CSV):

Amplication Plots

Melt Curves


Prx6

qPCR Report (PDF):

qPCR File (PCRD):

qPCR Data (CSV):

Amplication Plots

Melt Curves

Reverse Transcription – Ronit’s C.gigas DNased ctenidia RNA

Proceeded with reverse transcription of Ronit’s DNased ctenidia RNA (from 20181016).

Reverse transcription was performed using 100ng of each sample with M-MMLV Reverse Transcriptase from Promega.

Briefly, 100ng of DNased RNA was combined with oligo dT primers and brought up to a final volume of 15uL. Tubes were incubated for 5mins at 70oC in a PTC-200 thermal cycler (MJ Research), using a heated lid. Samples were immediately placed on ice.

A master mix of buffer, dNTPs, water, and M-MMLV reverse transcriptase was made, 10uL of the master mix was added to each sample, and mixed via finger flicking. Samples were incubated for 1hr at 42oC in a PTC-200 thermal cycler (MJ Research), using a heated lid, followed by a 5min incubation at 65oC.

Samples were stored on ice for use later this afternoon by Ronit.

Samples will be stored in Ronit’s -20oC box.

Reverse transcription calcs (Google Sheet):

qPCR – Ronit’s DNAsed C.gigas Ploidy/Dessication RNA with elongation factor primers

After I figured out the appropriate DNA and primers to use to detect gDNA in Crassostrea gigas samples, I checked Ronit’s DNased ctenidia RNA (from 20181016) for residual gDNA.

Elongation factor primers:

  • EF1_qPCR_5′ (SRID 309)
  • EF1_qPCR_3′ (SRID 310)

BB16 from 20090519 was used as a positive control.

Samples were run on Roberts Lab CFX Connect (BioRad). All samples were run in duplicate. See qPCR Report (Results section) for plate layout, cycling params, etc.

qPCR master mix calcs (Google Sheet):


Results

qPCR Report (PDF):

qPCR File (PCRD):

qPCR Data (CSV):

In the plots below, green is the positive control, blue are the samples, and red is the no template control (NTC).

Everything looks great! Nice, clean, gDNA-free RNA! Will proceed with reverse transcription.


Amplification Plots


Melt Curves

qPCR – C.gigas primer and gDNA tests with 18s and EF1 primers

The [qPCR I ran earlier today to check for residual gDNA in Ronit's DNased RNA] turned out terribly, due to a combination of bad primers and, possibly, bad gDNA.

I tracked down some different primers for testing:

  • Cg_18s_1644_F (SRID 1168)
  • Cg_18s_1750_R (SRID 1169)
  • EF1_qPCR_5′ (SRID 309)
  • EF1_qPCR_3′ (SRID 310)

In addition to BB15 from 20090519, I decided to test out BB16 from 20090519 as a positive control.

Samples were run on Roberts Lab CFX Connect (BioRad). All samples were run in duplicate. See qPCR Report (Results section) for plate layout, cycling params, etc.

qPCR master mix calcs (Google Sheet):


Results

qPCR Report (PDF):

qPCR File (PCRD):

qPCR Data (CSV):

Looks like the elongation factor (EF1) primers and BB16 gDNA as a positive control are the way to go.

In the plots below, the black lines are BB16, the green lines are BB15, and the red lines are no template controls (NTC).

The amplification plots show that the EF1 primers do not amplify with BB15, but do amplify with BB16 (black lines Cq ~34). The 18s primers amplify with both BB15 & BB16 (Cq ~16 & ~18, respecitively), but produce primer dimers (red lines in amplification and melt curve plots).


Amplification Plots


Melt Curves

qPCR – Ronit’s DNAsed C.gigas Ploidy/Dessication RNA with 18s primers

After DNasing Ronit’s RNA earlier today, I needed to check for any residual gDNA.

Identified some old, old C.gigas 18s primers that should amplify gDNA:

  • gigas18s_fw (SRID 157)
  • gigas18s_rv (SRID 156)

Used some old C.gigas gDNA (BB15 from 20090519) as a positive control.

Samples were run on Roberts Lab CFX Connect (BioRad). All samples were run in duplicate. See qPCR Report (Results section) for plate layout, cycling params, etc.

qPCR master mix calcs (Google Sheet):


Results

qPCR Report (PDF):

qPCR File (PCRD):

qPCR Data (CSV):

Well, this primer set and/or the gDNA is not good. In the plots below, the positive control gNDA is in green, samples in blue, and no template controls (NTC) are in red.

Poor performance is most easily noticed when looking at the melt curves. They have multiple peaks, suggesting non-specific amplification, even in the positive control.

Additionally, although less evident from just looking at the plots, is the replicates are highly inconsistent. Although it’s possible that might be due to poor technique, it’s very unlikely.

Will have to identify different primers and/or positive control DNA.


Amplification Plots


Melt Curves

DNase Treatment – Ronit’s C.gigas Ploiyd/Dessication Ctenidia RNA

After quantifying Ronit’s RNA earlier today, I DNased them using the Turbo DNA-free Kit (Ambion), according to the manufacturer’s standard protocol.

Used 1000ng of RNA in a 50uL reaction in a 0.5mL thin-walled snap cap tube. Samples were mixed by finger flicking and then incubated 30mins @ 37oC in a PTC-200 thermal cylcer (MJ Research), without a heated lid.

DNase inactivation was performed (0.1 volumes of inactivation reagent; 5uL), pelleted, and supe transferred to new 1.7mL snap cap tube.

Samples were stored on ice in preparation for qPCR to test for residual gDNA.

DNase calculations are here:

Samples will be permanently stored here (Google Sheet):

RNA Quantification – Ronit’s C.gigas Ploidy/Dessication RNA

Last Friday, Ronit quantified 1:10 dilutions of the RNA I isolated on 20181003 and the RNA he finished isolating on 20181011, but two of the samples (D11-C, T10-C) were still too concentrated.

I made 1:20 dilutions (1uL RNA in 19uL 0.1% DEPC-treated H2O) and quantified them using the Roberts Lab Qubit 3.0, with the RNA HS assay. Used 1uL of the diluted RNA.


RESULTS

Qubit data (Google Sheet):

Everything looks good. Added final concentration values (Qubit data x 20, to account for dilution factor) to Ronit’s master sheet (Google Sheet):

Will proceed with DNasing.

RNA Isolation – Ronit’s C.gigas diploid/triploid dessication/heat shock ctenidia tissues

Isolated RNA from a subset of Ronit’s Crassostrea gigas ctenidia samples (see Ronit’s notebook for experiment deets):

  • D01 C

  • D02 C

  • D19 C

  • D20 C

  • T01 C

  • T02 C

  • T19 C

  • T20 C

RNA was isolated using RNAzol RT (Molecular Research Center) in the following way:

  • Unweighed, frozen tissue transferred to 500uL of RNAzol RT and immediately homogenized with disposable pestle.

  • Added additional 500uL of RNAzol RT and vortexed to mix.

  • Added 400uL of 0.1% DEPC-treated H2O, vortexed and incubated 15mins at RT.

  • Centrifuged 12,000g for 15mins at RT.

  • Transferred 750uL of supernatant to clean tube (discarded remainder), added 1 volume (750uL) of isopropanol, vortexed, and incubated at RT for 10mins.

  • Centrifuged 12,000g for 10mins at RT.

  • Discarded supernatant.

  • Washed pellet with 75% ethanol (made with 0.1% DEPC-treated H2O).

  • Centrifuged 4,000g for 2mins at RT.

  • Discarded supernatant and repeated wash steps.

Pellet was resuspended in 50uL of 0.1% DEPC-treated H2O and stored @ -80oC in Ronit’s temporary box.

Samples Received – Triploid Crassostrea gigas from Nisbet Oyster Company

Received a bag of Pacific oysters from Nisbet Oyster Company.

Four oysters were shucked and the following tissues were collected from each:

  • ctenidia
  • gonad
  • mantle
  • muscle

Utensils were cleaned and sterilized in a 10% bleach solution between oysters.

Tissues were stored briefly on wet ice and then stored at -80C in Rack 2, Column 3, Row 1

Dissection – Frozen Geoduck & Pacific Oyster

We’re working on a project with Washington Department of Natural Resources’ (DNR) Micah Horwith to identify potential proteomic biomarkers in geoduck (Panopea generosa) and  Pacific oyster (Crassostrea gigas). One aspect of the project is how to best conduct sampling of juvenile geoduck (Panopea generosa) and Pacific oyster (Crassostrea gigas) to minimize changes in the proteome of ctenidia tissue during sampling. Generally, live animals are shucked, tissue dissected, and then the tissue is “snap” frozen. However, Micah’s crew will be collecting animals from wild sites around Puget Sound and, because of the remote locations and the means of collection, will have limited tools and time to perform this type of sampling. Time is a significant component that will have great impact on proteomic status in each individual.

As such, Micah and crew wanted to try out a different means of sampling that would help preserve the state of the proteome at the time of collection. Micah and crew have collected some juveniles of both species and “snap” frozen them in the field in a dry ice/ethanol bath in hopes of being able to best preserve the ctenidia proteome status. I’m attempting to dissect out the frozen ctenidia tissue from both types of animals and am reporting on the success/failure of this method of preservation-sampling protocol.

To test this, I transferred animals (contained in baggies) from the -80C to dry ice. Utensils and weigh boats were cooled on dry ice.

 

Results:

Quick summary: This method won’t and I think sampling will have to take place in the field.

The details of why this won’t work (along with images of the process) are below.

 

First issue with this sampling method (and should be noted because I believe dry ice/ethanol baths will be used, even with a different sampling methodology) is that the ethanol in the dry ice bath at the time of animal collection is a potential problem for labeling baggies. Notice in the screenshot below that the label for the geoduck baggie (the baggie on the left) has, for all intents and purposes, completely washed off:

 

 

Starting with C.gigas, opening the animal was relatively easy. Granted, the animal has become brittle, but access to, and identification of, tissues ended up being pretty easy:

 

 

 

 

However, dissecting out just ctenidia is a lost cause. The mantle and the ctendia are, essentially, fused together in a frozen block through the oyster. Although the image below might look like part of the shell, it is not. It is strictly a chunk of frozen ctenidia/mantle tissue:

 

 

 

The geoduck were even more difficult. In fact, I couldn’t even manage to remove the soft tissue from the shell (for the uninitiated, there are two geoduck in the image below). I only managed to crush most of the tissue contained within the shell, making it even more impossible (if that’s possible) to identify and dissect out the ctenidia: