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). 0.5uL (~40ng) 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:
Residual gDNA is present in the sample. So, it’s become apparent that it’s virtually impossible to rid the BB01 RNA of contaminating gDNA. Will discuss with Steven and Mac if it’s feasible to exclude this from the additional PROPS analysis that needs to be done and how this could potentially affect our data. Talked to Steven and, duh, we can just remove the previous BB01 data from our analysis. Will make new batch of cDNA from existing DNased RNA samples.
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.
All runs (3 runs were conducted) were created using a master mix containing C.gigas gDNA (either 50ng or 100ng), 1X Promega qPCR Master Mix, 0.2uM each of forward/reverse primers (18s; Roberts SR ID: 156, 157). The master mix was mixed well and 10uL were distributed in each well of ABI plates. Plates were sealed with ABI optical adhesive covers.
It should also be noted that this analysis was only done with a single primer set and was not tested on any other qPCR machines. This can easily be done if it is desired, however I think one of the issues still being observed with the machine is sample-independent (see Results section below).
Results:
Here’s an extremely quick and dirty analysis of what these qPCR runs have revealed (across the entire plate, 3 plates of data):
Avg. Range of Cts Across Plates – 1.70
Avg. Std. Deviation of Cts Across Plates – 0.352
Based off of the graphs below (particularly the Ct vs Well Position plot), my conclusion is that the machine reads plates inaccurately in Rows A, B, C, F, G, & H. Rows D & E exhibit the most consistent well-to-well readings and, potentially, could be used for qPCR.
The entire work up (which includes a breakdown of each well position relative to each other) is here (Excel Workbook .xlsx). Below are screen captures of one of the three plates (as an example, since all looked the same) that were used for analysis of the amplification plots, melt curves and Ct vs Well Position and a quick description/assessment of what I have observed.
The amplification plot (below) clearly shows the type of spread in Cts across an entire plate that was observed in each run, as well as a large range in fluorescence detected (Rn) in each well.
The melt curve (below) reflects the large range of detected fluorescence seen in the amplification plot. Additionally, some wells exhibit small “bumps” between 75C and 80C. This provides more evidence for a problem with well-to-well consistency.
A graph of Ct vs. Well Position (below) reveals some enlightening information. From looking at this plot, it’s clear that the machine reads from A1 to A12, then B1 to B12 (reads by row, not column) and so on. This plot reveals that most of the variation seen in Ct values occurs in the two rows closest to the edge of the plate, and within those rows, the middle wells’ Cts are more similar to the Cts observed throughout the rest of the plate.
This is a repeat of the two runs from yesterday, just to see if there is a correlation between the failed plates being the first of the day or not. Master mix calcs and cycling params are here (these calcs are from yesterday, but were used again for today).
Results:
Amplification in all wells. Still seeing ~3 cycle spread across the entire plate. Will work up all three successful sets of run data.
This was repeated from earlier today due to the failure of the previous run, but had to use new gDNA since I ran out of the stock I had previously used. Master mix calcs and cycling params are here.
Results:
Amplification in all wells, however well E4 appears to have had some evaporation (and the effects can clearly be seen in the amplification plot below). Still getting ~3 cycle spread across the entire plate, which is disconcerting. Oddly, this is the second day where the 1st run completely failed, but the 2nd run was successful…
This is a repeat of a run from 20110204. Here’re master mix calcs. This was being repeated to evaluate whether or not the relative differences in Ct values observed on 20110204 are consistent or not across the plate. Cycling params were as follows:
40 Cycles of:
Melt curve.
Results:
Absolutely no amplification of any kind! Bizarre. Will repeat.
This is a repeat of an earlier run from today, but with a different qPCR plate. Here’re master mix calcs (bottom half of page). Cycling params were as follows:
40 Cycles of:
Melt curve.
Results:
All samples amplified and showed a proper dissociation curve. However, it does look like there’s a spread of ~3 Cts across the plate. This is not good, as this is the equivalent of ~10-fold difference in gene expression. Will repeat again and see if specific wells show the same relative differences in Cts.
