Performed qPCR using cDNA from 20110311. This was performed for additional reps for TIMP3(BB) (SR IDs:1067 & 1106) and HMGP (SR IDs:359 & 360). Master mix calcs are here. Plate layout, cycling params, etc can be found in the qPCR Report (see Results).
Results:
Will analyze with PCR Miner and incorporate with previous PCR rep done for PROPS with these two genes. Oddly, samples in wells B09 and H09 have weird melt curves. As such, these samples will be excluded from analysis.
Ran qPCR on DNased RNA from earlier today to verify removal of contaminating gDNA. Used C.gigas 18s primers (SR IDs: 156, 157). 0.75uL (~50ng) of DNased RNA was used for testing. This corresponds, roughly, to the amount of sample that would be carried through to qPCR analysis of cDNA, assuming 1ug of RNA was used to make the cDNA (cDNA = 1000ng RNA/25uL = 40ng/uL, 1uL of cDNA in 25uL qPCR reaction). Positive control sample was ~25ng BB16 gDNA (from 20090519). Master mix calcs are here. Plate layout, cycling params, etc can be found in the qPCR Report (see Results). RNA was stored @ -80C in “Sam’s -80C Box”.
Results:
Well, this sucks. Still gDNA contamination. Will just start with original RNA again and discard this “DNased” sample.
Ran qPCR on DNased RNA from earlier today to verify removal of contaminating gDNA. Used C.gigas 18s primers (SR IDs: 156, 157). 0.5uL (50ng) of DNased RNA was used for testing. This corresponds, roughly, to the amount of sample that would be carried through to qPCR analysis of cDNA, assuming 1ug of RNA was used to make the cDNA (cDNA = 1000ng RNA/25uL = 40ng/uL, 1uL of cDNA in 25uL qPCR reaction). Positive control sample was ~25ng BB16 gDNA (from 20090519). Master mix calcs are here. Plate layout, cycling params, etc can be found in the qPCR Report (see Results). RNA was stored @ -80C in “Sam’s -80C Box”.
Results:
Ugh. Still gDNA present in this sample. Hmmmm. Will consider starting from original RNA, but will precipitate this sample again and treat again to see if I can get rid of that cursed gDNA.
Ran qPCR on DNased RNA from earlier today to verify that it was free of contaminating gDNA. Used C.gigas 18s primers (SR IDs: 156, 157). 0.5uL (50ng) of DNased RNA was used for testing. This corresponds, roughly, to the amount of sample that would be carried through to qPCR analysis of cDNA, assuming 1ug of RNA was used to make the cDNA (cDNA = 1000ng RNA/25uL = 40ng/uL, 1uL of cDNA in 25uL qPCR reaction). Positive control sample was ~25ng BB16 gDNA (from 20090519). Master mix calcs are here. Plate layout, cycling params, etc can be found in the qPCR Report (see Results). RNA was stored @ -80C in “Sam’s -80C Box”.
Results:
There is residual gDNA in the BB01 sample. Will EtOH precipitate and treat again.
DNased BB09 was stored @ -80C in “Mac’s Gigas DNased RNA Box #1″ (on the top shelf) with the rest of the PROPS DNased RNA.
Used new cyclooxygenase primers (SR IDs 1060, 1061) to see how they performed and to evaluate tissue distribution. Tissue distribution was evaluated using the following cDNAs made on 10/27/10 from Emma:
Gigas Digestive Gland
Gigas Gill
Gigas Mantle
Gigas Muscle
qPCR Master Mix calcs are here. Plate layout, cycling parameters, etc can be found in the qPCR Report (see Results).
Results:
qPCR Report (PDF).
Amplification is present in all four tissue types and the melting curve looks good. So, these primers are good to go. Steven suggests checking to see if we see a change in gene expression from an old experiment of Gigas exposed to high levels of Vibrio tubiashii. Will round up some old cDNA for this.
Performed qPCR on Friedman Lab machine targeting immune-related genes in hard clam. Rough plate layout/master mix calcs are here. qPCR report from Friedman Lab machine is here (PDF) and shows cycling params, plate layout and Cts.
Results:
The following primer sets failed to produce an amplicon:
Mm_TRAF6
Mercenaria_Rel
TLR
STI
CytP450-like
Raw fluorescence data was extracted (No baseline subtraction) and processed with PCR Miner. Data workup/analysis is here. Here is a graph of those primer sets producing an amplicon. All were normalized to actin, which exhibited the smallest amount of deviation across all three samples of the normalizing/housekeeping genes analyzed.
As a preliminary run with these genes, there are a number of promising candidates that could yield some interesting data regarding the physiological response of hard clam to exposure to QPX.
