qPCRs – Tim’s adult gigas challenge cDNA (from 20091009)

Set up qPCR with Cg_P450 primers and TNFRAF3’/5′ primers. Plate layout/setup is here.

Results: Processed with PCR Miner. Normalized to EF1. Standard Error bars. Here is spreadsheet with workup.

P450: The two control samples (AC & CC) show significant difference in expression between the Air and CO2 treated samples. The Vibrio treated samples (AV & CV) show no difference in expression between Air and CO2 treatments.

The Air samples (AC & AV) show significant difference in expression between the Control (AC) and Vibrio treated (AV) samples.

TNFRAF3: Expression levels of the Vibrio treated samples (AV &CV) were too low for Miner processing.

There are significant differences in expression between the Air (AC) and CO2 treated (CC) samples.

 

Set up qPCR with Cg_IkB primers and Cg_Prx6 primers. Plate layout/setup is here.

Results: Processed with PCR Miner. Normalized to EF1. Standard Error bars. Here is spreadsheet with workup.

IkB:

No difference in expression between Air (AC & AV) and CO2 (CC & CV) treatments.

Appears to be a significant difference between the Air Control (AC) and the Air Vibrio treated (AV) samples.

Prx6:

Significant difference in expression between Air Control (AC) and CO2 Control (CC) samples, but no difference in the Vibrio treated samples.

Significant difference in expression between the Air Control (AC) and the Air Vibrio treated (AV) samples, but no difference in the CO2 treated Vibrio samples (CC & CV).

qPCR – Tim’s adult gigas challenge cDNA (from 20091009)

Set up qPCR with Cg_HIF1 (hypoxia induced factor 1) primers and prostaglandin E2 primers. Plate layout/setup is here.

Results: Processed with PCR Miner. Normalized to EF1. Standard Error bars. Here is spreadsheet with workup.

HIF1:

No significant differences between any treatments.

Prostaglandin E2:

No significant differences between any treatments.

qPCR – Tim’s adult gigas challenge cDNA (from today)

Set up qPCR with EF1 primers and IL17 Internal primers. Plate layout/setup is here. Note: gDNA sample used as a “positive” control will NOT amplify with the EF1 primers.

Results: Processed with PCR Miner. Normalized to EF1. Standard Error bars. Here is spreadsheet with workup.

IL17 Internal:

No significant differences between any treatments.

qPCR – Tim’s adults gigas challenge re-DNased RNA (from today)

Performed qPCR using q18s primers on re-DNased RNA (1:100 dilution to match final concentration of template after making cDNA). qPCR set up and plate layout are here.

gDNA dilutions were used as positive controls. gDNA = BB11 (0.49ug/uL) from 20090519. Used 5uL of 1:10, 1:100 and 1:000 dilutions.

Results: re-DNase-ing the samples seems to have worked. Positive controls are the only samples to come up. Will proceed to making cDNA.

qPCR – Tim’s adults gigas challenge DNased RNA (from today)

Previous qPCR was done incorrectly (wrong primers), so am repeating with the correct primers. Performed qPCR using q18s primers on DNased RNA (1:100 dilution to match final concentration of template after making cDNA). qPCR set up and plate layout are here.

gDNA dilutions were used as positive controls. gDNA = BB11 (0.49ug/uL) from 20090519. Used 5uL of 1:10, 1:100 and 1:000 dilutions.

Results: Still had 5 samples that came up positive. Will re-DNase treat these samples and then re-qPCR them.

qPCR – Tim’s adults gigas challenge DNased RNA (from today)

Performed qPCR using q18s primers on DNased RNA (1:100 dilution to match final concentration of template after making cDNA). qPCR set up and plate layout are here.

gDNA dilutions were used as positive controls. gDNA = BB11 (0.49ug/uL) from 20090519. Used 5uL of 1:10, 1:100 and 1:000 dilutions.

Results: All samples, including positive controls, came up negative! Realized that I accidentally used the EF1 primers which will NOT amplify gDNA. And, to top it off, this was BEFORE going to seminar (i.e. before having any beers).

DNase Treatment – Tim’s adult gigas challenge RNA (from 20090930)

Used 5uL of RNA from each sample, brought samples up to 50uL with H2O and treated according to Ambion’s Turbo DNA-free kit. Rigorous protocol was followed (1.5uL DNase initially + 1.5uL additional DNase after 30mins). Transferred treated samples to a PCR plate to facilitate further manipulation of the samples. Will perform qPCR on these samples to make sure treatment worked.

Bioanalyzer Submission – Trout RBC, Colleen’s gigas GE sample, Mac’s DH/BB PCR for SOLiD WTK

Samples were delivered for analysis on the DNA 1000 chip.

UPDATE 20091008 They do not have the DNA 1000 kit in stock. Will be using High Sensitivity Kit instead. Will have in stock on Tuesday.

Results: