Tag Archives: Venerupis philippinarum

qPCR – Manila Clam Larvae cDNA (from August 2012 – Dave’s Notebook)

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.

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qPCR – Manila Clam Larvae cDNA (from August 2012 – Dave’s Notebook)

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_Calmodulin_F/R2 (SR IDs: 1449, 1467)

Rp_Crumbs_F/R (SR IDs: 1477, 1476)

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_161738.tad

qPCR Raw Data and PCR Miner Analysis (Excel) http://eagle.fish.washington.edu/Arabidopsis/qPCR/Opticon/Sam_20121108_161738.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 later today.

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qPCR – Manila Clam Larvae cDNA (from August 2012 – Dave’s Notebook)

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_GPX3_F/R2 (SR IDs: 1453, 1469)

Rp_HSP90_F2/R2 (SR Ids: 1457, 1471)

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_150936.tad

qPCR Raw Data and PCR Miner Analysis(Excel) http://eagle.fish.washington.edu/Arabidopsis/qPCR/Opticon/Sam_20121108_150936.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 later today.

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qPCR – DNased Manila Clam Larvae RNA (from August 2012 – Dave’s Notebook)

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

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Reverse Transcription – Dave’s Manila Clam (Venerupis philippinarum) DNased RNA from 20120307 and 20120302

Performed reverse transcription on 1.5ug of DNased RNA in a 75uL reaction, using oligo dT primers. All reagents were scaled appropriately (based on Promega’s M-MLV RT protocol). Samples were prepared in a plate and stored @ -20C. Plate layout and all reverse transcription calcs are here:

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qPCR – Dave’s Manila Calm (Venerupis philippinarum) DNased RNA from yesterday and 20120302

Performed qPCR on all DNased RNA samples from this group (samples #1-48) using beta actin primers (SR IDs: 1379, 1380). 0.5uL of each DNased RNA was used, which was the equivalent of ~40ng, in order to simulate the amount of RNA present in the subsequent cDNA (1000ng of RNA in 25uL cDNA; use 1uL of cDNA in qPCR reaction). Master mix calcs are here. Plate layout, cycling params, etc., can be found in the qPCR Report (see Results). 0.5uL of total RNA from sample Vp gill 01 was used to serve as a positive control, since Dave has no existing V. phillippinarum cDNA.

Results:

qPCR Data File (CFX96)

qPCR Report (PDF)

All samples are clean and are ready for reverse transcription.

Of note, the overall fluorescence of the reactions was very low. As such, the default baseline analysis setting (linear) suggested that all samples had a Cq value because the baseline was incorrectly set an was NOT above background fluorescence levels. Changing the baseline analysis setting to “regression” resolved this. Also, it should be noted that one sample (#48) other than the positive control actually does show amplification and a corresponding melt curve. However, the melt curve peak is at a different temp than the positive control, suggesting that this is non-specific amplification in sample #48.

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RNA Isolation – Dave’s Manila Clam (Venerupis philippinarum) Gill Samples (#25-48)

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:

All samples look great with excellent yields and great 260/280 values. Will proceed with DNasing. (Note: Sample #42 appears twice because the first reading had an air bubble and, as such, should be discarded.)

 

DNased RNA using Ambion’s Turbo DNA-free Kit following the “routine” protocol. 5ug of total RNA from each sample was treated in 50uL reactions. Samples will be spec’d on Monday with the Roberts Lab NanoDrop 1000.

Results:

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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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