qPCR – Check DNased RNA BB01 for Residual gDNA (from earlier today)

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:

qPCR Report (PDF)

qPCR Data File (CFX96)

Well, this sucks. Still gDNA contamination. Will just start with original RNA again and discard this “DNased” sample.

DNase – C.gigas BB01 from 20110225

Used EtOH precipitated BB01 RNA from 20110225 and followed Ambion’s “rigorous” protocol, utilizing a total of 2uL of DNAse. Briefly, samples were incubated @ 37C for 30mins, an additional 1uL of DNase was added to each sample, mixed and incubated for an additional 30mins @ 37C. After finishing protocol, samples were spec’d.

Results:

RNA looks good, based on the OD260/280. As usual after DNasing, the OD260/230 is on the low side. Will check for residual gDNA via qPCR.

Ethanol Precpitation – DNased RNA BB01 (from earlier today)

Due to residual gDNA contamination, will EtOH precipitate in order to treat with DNase again. Add 0.5 vols 3M NaAOc (pH=

5.2), 2.5 vols 100% EtOH, mixed and incubated @ -20C for 30mins. Pelleted RNA @ 16,000g, 4C 30mins. Washed RNA with 1mL 70% EtOH (2x due to fear of residual salts from DNase Buffer). Pelleted RNA @ 16,000g, 4C, 15mins. Resuspended RNA in 45uL nuclease-free H2O. Sample was stored @ -80C (in “Sam’s RNA Box #1) until it could be DNased again.

qPCR – Check DNased RNA BB01 for Residual gDNA (from earlier today)

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:

qPCR Report (PDF)

qPCR Data File (CFX96)

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.

DNase – C.gigas BB01 from 20110216

Used EtOH precipitated BB01 RNA from 20110216 and followed Ambion’s “rigorous” protocol, utilizing a total of 2uL of DNAse. Briefly, samples were incubated @ 37C for 30mins, an additional 1uL of DNase was added to each sample, mixed and incubated for an additional 30mins @ 37C. After finishing protocol, samples were spec’d.

Results:

First reading had an air bubble and should be ignored. DNased RNA looks good, based on 260/280 ratios. As is usually the case for DNased RNA, the 260/230 ratios are on the low side. Will check DNased RNA for residual gDNA.

Ethanol Precipitation – DNased RNA BB01 (from earlier today)

Due to residual gDNA contamination, will EtOH precipitate in order to treat with DNase again. Add 0.5 vols 3M NaAOc (pH = 5.2), 2.5 vols 100% EtOH, mixed and incubated @ -20C for 30mins. Pelleted RNA @ 16,000g, 4C 30mins. Washed RNA with 1mL 70% EtOH (2x due to fear of residual salts from DNase Buffer). Pelleted RNA @ 16,000g, 4C, 15mins. Resuspended RNA in 45uL nuclease-free H2O. Sample was stored @ -80C (in “Sam’s RNA Box #1) until it could be DNased again.

qPCR – Check DNased RNA BB01 & 09 (from earlier today)

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:

qPCR Report (PDF).

qPCR Data file (CFX96).

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.

DNase – C.gigas BB/DH (PROPS) RNA (from 20090507)

Need more DNased RNA to finish repeating of PROPS. Some samples had insufficient quantities of DNased RNA remaining in BB01 and BB09. Used 10ug of each RNA and followed Ambion’s “rigorous” protocol, utilizing a total of 2uL of DNAse for each sample. Briefly, samples were incubated @ 37C for 30mins, an additional 1uL of DNase was added to each sample, mixed and incubated for an additional 30mins @ 37C. After finishing protocol, samples were spec’d.

DNase Rxn Calcs:

BB01 (1.824ug/uL): 10ug/1.824ug/uL = 5.48uL RNA + 39.52uL H2O (to 45uL) + 5uL 10X DNase Buffer = 50uL

BB09 (0.506ug/uL): 10ug/0.506ug/uL = 19.77uL RNA + 25.23uL H2O (to 45uL) + 5uL 10X DNase Buffer = 50uL

Results:

260/280 values look great. 260/230 values look bad, but this is not unusual for samples post-DNase treatment.

Data Analysis – Young Lab ABI 7300 Calibration Checks

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.

qPCR – Test Young Lab qPCR Calibration

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.