Ran qPCR on manila clam larvae cDNA that Dave made on 8/7/2012, using the sample sets from 7/29/2011 and 8/5/2011 of the OA manila clam experiment he ran.

Primers used:

Rp_Cathepsin_F/R2 (SR IDs: 1461, 1473)

Rp_EF1a_F/R2 (SR IDs: 1463, 1474)

Primers were verified to be in good working order by Dave on 4/1/2012 (see Dave’s notebook).

Master mix calcs are here. Cycling params can be found in the qPCR Data File (see Results). Plate layout and PCR Miner analysis can be found in the qPCR Raw Data file (see Results). All samples run in duplicate.

Results:

qPCR Data File(Opticon 2) http://eagle.fish.washington.edu/Arabidopsis/qPCR/Opticon/Sam_20121108_172259.tad

qPCR Raw Dat and PCR Miner Analysis(Excel) http://eagle.fish.washington.edu/Arabidopsis/qPCR/Opticon/Sam_20121108_172259.xlsx

Reps look pretty good, although the 4C2 8.5.11 sample has consistently bad reps across all of today’s runs. Data will be shared with Steven for comparison to Dave’s Illumina data.

All data was normalized to EF1a expression from this run.

Performed qPCR on Dave’s manila clam larvae DNased RNA from August 2012 using EF1a primers (SR IDs: 1463, 1474).

Master mix calcs are here. https://docs.google.com/spreadsheet/ccc?key=0AmS_90rPaQMzdHc5amwzZzdDa1d0VXQzLVU0WkFTc0E

Plate layout, cycling params, etc can be found in the qPCR Report (see Results).

Positive control was pooled cDNA taken from Dave’s cDNA plate on 8/7/2012.

Results:

qPCR Data File(CFX96) http://eagle.fish.washington.edu/Arabidopsis/qPCR/CFX96/Roberts%20Lab_2012-10-26%2010-48-07_CC009827.pcrd

qPCR Report(PDF) http://eagle.fish.washington.edu/Arabidopsis/qPCR/CFX96/Roberts%20Lab_2012-10-26%2010-48-07_CC009827.pdf

Here’s a quick Google Spreadsheet summary highlighting samples that came up positive/negative.

https://docs.google.com/spreadsheet/ccc?key=0AmS_90rPaQMzdFFHb3YwWE01UG00TnY3OWo2cWx2UVE

Approximately half of the samples (~27) came up positive for still having gDNA in them.

There are three pCO2 treatments: 1000ppm, 750ppm, and 400ppm. There are six sampling dates: 7/29/2011, 8/2/2011, 8/9/2011, 8/12/2011. Currently, it is unknown when the Day 0 samples were collected. Have emailed Dave for deets.

There are only two dates (7/29/2011 and 8/5/2011) that have a full set of samples (i.e. 1000ppm, 750ppm and 400ppm) that exhibit DNA-free RNA. Will discuss with Steven on how to proceed.

UPDATE 20121031 – Dave emailed and indicated the experimented started on 7/27/2011. Additionally, the two sample sets that are complete are Day 2 and Day 7. Discussing with Steven, we have decided to run a few genes and see how the expression levels compare to the NGS data analysis for these samples. If the qPCR data supports the NGS data, then that information will be relayed to the BMC Genomics reviewers in response to their critiques. A copy of the manuscript is here(may not be publicly viewable). https://docs.google.com/document/d/1Ii1lODz2oThiyxZtHBblUEdzyhIVq92n8jkEjhkuuts/edit

Performed qPCR on all 12 samples. Used Cg_EF1aF/R2 (SR IDs: 1410 & 1412) for one set of qPCRs and Vtub_16s_F/R (SR IDs: 455 & 456) for the other set of qPCRs. Used pooled C.gigas cDNA (from 20110311) and RE22 DNA (provided by Elene) as positive controls for C.gigas and V.tubiashii, respectively. C.gigas gDNA (7ng of BB16 from 20110201) was used as a negative control for EF1a. Master mix calcs are here. Plate layout, cycling params, etc can be found in the qPCR Report (see Results). All samples were run in duplicate.

Results:

qPCR Data File (CFX96)

qPCR Report (PDF)

C.gigas EF1a – Positive control amplified. Negative control and no template control were all clean (i.e. no amplification detected). The majority of samples had amplification, however two samples had no amplification at all (samples 132 & 136).

V.tubiashii 16s – Positive control amplified. No template controls exhibited amplification in both replicates. All samples exhibited amplifcation, however nearly all of the melt curves have multiple peaks present, suggesting that more than one target is being amplified. I suspect this is due to residual gDNA, but this fails to explain the amplification in the no template controls which also exhibited dual peaks in the melt curves.

Spoke with Steven and he suggested to skip troubleshooting the V. tubiashii 16s for now and proceed with trying to qPCR some additional V.tubiashii genes. Will talk with Elene to see if/which additional genes she has primers for.

Ran a qPCR on all cDNA samples from the V.vulnificus exposure experiment from 20110111. This qPCR was to test 2 of 4 potential normalizing genes to evaluate which genes show the least amount of effect from the treatments in this experiment. Primers for 18s used were Cg_18s_1644_F (SR ID: 1168), Cg_18s_1750_R (SR ID: 1169). Primers for EF1a used were EF1_qPCR_5′ (SR ID: 309), EF1_qPCR_3′ (SR ID: 310)Samples were run in duplicate. Master mix calcs are here. The master mix info is the same that was used earlier today, but with the primers noted above, not those listed on the calcs page. Plate layout, cycling params, etc., can be seen in the qPCR Report (see Results).

Results:

qPCR Data File (BioRad CFX96)

qPCR Report (PDF)

18s: Average Cq = 22.39, Standard Deviation = 0.905

EF1a: Average Cq = 20.59, Standard Deviation = 0.658

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.