Tag Archives: qPCR

qPCR – Check DNased RNA from Earlier Today for Residual gDNA

Ran qPCR using V.tubiashii VtpA primers (from Elene; no SR ID). Used 0.5uL of each DNased RNA sample, which equals ~40ng of RNA, which would be the equivalent amount of RNA that would end up in a qPCR rxn after cDNA has been made (using 1uL of cDNA). Used the filter DNA extraction from samples #279 from DATE as a positive control. Master mix calcs are here. Plate layout, cycling params, etc. can be found in the qPCR Report (see Results).

Results:

qPCR Data File (CFX96)

qPCR Report (PDF)

All samples showed up negative, except for the positive control. Will proceed with making cDNA on Monday.

qPCR – Taylor Water Filter DNA Extracts from 20120322

Ran qPCR on the Taylor water filter DNA extracts from 20120322 using V.tubiashii VtpA primers (provide by Elene; no SR ID?) instead of 16s primers, which failed to produce acceptable results in the melt curves (see 20120323). Additionally, Elene has a standard curve for V. tubiashii (from 1/12/2011) based off of CFUs/mL, which will allow us to quantify theoretical number of V.tubiashii CFUs present in each sample.

Master mix calcs are here. Plate layout, cycling params, etc. can be found in the qPCR Report (see Results).

Results:

qPCR Date File (CFX96)

qPCR Report (PDF)

Overall, the run looks excellent. Both negative controls and no template controls are clean. Since I was able to use a standard curve, I determined CFUs of V.tubiashii in each sample, as follows:

Mean CFUs per qPCR reaction / template volume per qPCR reaction x filter extraction elution volume (100uL) = total CFUs on water filter.

Total CFUs on filter / filtered water volume = CFUs per mL in Taylor tanks

158 – 16500 copies/2uL = 8250 copies/uL x 100uL = 825000 copies on water filter/1000mL = 825 copies/mL

200 – 5700 copies/2uL = 2850 copies/uL x 100uL = 285000 copies on water filter/1000mL = 285 copies/mL

279 – 325000 copies/2uL = 162500 copies/uL x 100uL = 16250000 copies on water filter/1000mL = 16250 copies/mL

313 – 152 copies/2uL = 76 copies/uL x 100uL = 7600 copies on water filter/1000mL = 7.6 copies/mL

341 – 124000/2uL = 62000 copies/uL x 100uL = 6200000 copies on water filter/1000mL = 6200 copies/mL

410 – 132000/2uL = 66000 copies/uL x 100uL = 6600000 copies on water filter/1000mL = 6600 copies/mL

433 – 63700/2uL = 31850 copies/uL x 100uL = 3185000 copies on water filter/1000mL = 3185 copies/mL

503 – 110/2uL = 55 copies/uL x 100uL = 5500 copies on water filter/1000mL = 5.5 copies/mL

551 – 2000/2uL = 1000 copies/uL x 100uL = 100000 copies on water filter/1000mL = 100 copies/mL

604 – 272/2uL = 136 copies/uL x 100uL = 13600 copies on water filter/1000mL = 13.6 copies/mL

Sample #410 was from the only tank that exhibited mortalities and was the only group of oyster larvae that showed any expression from the V.tubiashii genes (see DATE).

qPCR – Repeat of qPCR from Earlier Today

Repeated exactly what was done earlier today due to apparent contamination in negative controls.

Results:

qPCR Date File (CFX96)

qPCR Report (PDF)

Essentially the same results as the previous run. No template controls do amplify, but EXTREMELY weak and late. Melt curve analysis shows that the signals for the no template controls don’t cross the threshold set by the software.

However, I just looked back at the qPCR results from 20120208 where I used these V. tubiashii 16s primers and realized I got the same results from the cDNA (double-peaks in melt curves and amplification in the no template controls)!! So, I suspect that this primer set isn’t that useful. Will have to examine other sets of V. tubiashii 16s primers to use. Will discuss with Steven.

qPCR – Taylor Water Filter DNA Extracts from Yesterday

Ran qPCR on the Taylor water filter DNA extracts from yesterday using V.tubiashii 16s primers (SR IDs: 455, 456). Used RE22 DNA as a positive control, provided by Elene. Master mix calcs are here. All samples were run in duplicate. Plate layout, cycling params, etc can be found in the qPCR Report (see Results).

Results:

qPCR Data File (CFX96)

qPCR Report (PDF)

All samples amplified, including the negative controls. Negative controls exhibited very weak, late amplification. Additionally, many of the samples have a “shoulder” or apparent double-peak present in the melt curves. Will repeat to see if I can eliminate amplification in negative control samples.

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.

qPCR – cDNA from 20120208

Performed qPCR on all 12 samples. Used the following primers, provided by Elene, to detect V.tubiashii expression:

  • rseA_F/R
  • VtpA_F/R
  • VtpR_F/R

Used RE22 DNA (provided by Elene) as a positive control. Master mix calcs are the same as yesterday’s qPCR, but using the primers mentioned above. Plate layout, cycling params, etc. can be found in the qPCR Report (see Results). All samples were run in duplicate.

Results:

qPCR Data File (CFX96)

qPCR Report (PDF)

Positive control worked in all primer sets. All no template controls were clean for all primer sets.

Only one sample (#411) produced any amplification. Amplification was detected in the vtpA primer set (mean Cq = 38.06). However, there was also amplification detected in one of the two replicates for sample #411 in the rseA primer set (Cq = 39.09).

qPCR – cDNA from earlier today

Performed qPCR on all 12 samples. Used Cg_EF1aF/R2 (SR IDs: 1410 & 1412) for one set of qPCRs and Vtub_16s_F/R (SR IDs: 455 & 456) for the other set of qPCRs. Used pooled C.gigas cDNA (from 20110311) and RE22 DNA (provided by Elene) as positive controls for C.gigas and V.tubiashii, respectively. C.gigas gDNA (7ng of BB16 from 20110201) was used as a negative control for EF1a. Master mix calcs are here. Plate layout, cycling params, etc can be found in the qPCR Report (see Results). All samples were run in duplicate.

Results:

qPCR Data File (CFX96)

qPCR Report (PDF)

C.gigas EF1a – Positive control amplified. Negative control and no template control were all clean (i.e. no amplification detected). The majority of samples had amplification, however two samples had no amplification at all (samples 132 & 136).

V.tubiashii 16s – Positive control amplified. No template controls exhibited amplification in both replicates. All samples exhibited amplifcation, however nearly all of the melt curves have multiple peaks present, suggesting that more than one target is being amplified. I suspect this is due to residual gDNA, but this fails to explain the amplification in the no template controls which also exhibited dual peaks in the melt curves.

Spoke with Steven and he suggested to skip troubleshooting the V. tubiashii 16s for now and proceed with trying to qPCR some additional V.tubiashii genes. Will talk with Elene to see if/which additional genes she has primers for.

qPCR – DNased RNA from earlier today

Checked DNased RNA samples from earlier today for the presence of residual gDNA. Used C.gigas BB16 gDNA (from 20110201) diluted to ~7ng/uL as a positive control to match the dilution factor of the RNA that will be used in the reverse transcription reaction (175ng in 25uL = 7ng/uL). All samples were run in duplicate. Master mix calcs are here. Plate layout, cycling params, etc can be found in the qPCR Report (see Results).

Results:

qPCR Data (CFX96)

qPCR Report (PDF)

Positive control (in Green in qPCR Report) worked perfectly and showed excellent repeatability. The remainder of the samples (in Blue in qPCR Report) and the NTCs (in Red in qPCR Report) were extremely inconsistent with many having one replicate show late amplification, while the other replicate showed no amplification at all. Will have to repeat to get a more definitive assessment of residual gDNA content in these samples.

qPCR – C.gigas V.vulnificus Exposure cDNA (from 20110311)

Ran a qPCR using 3hr Vibrio vulnificus exposure cDNA from 20110311. Original experiment conducted on 20110111 with defensin primers (SR IDs: 1109 & 1070) and GAPDH (SR IDs: 1172 & 1173). Master mix calcs are here. Cycling params, plate layout, etc can be seen in the qPCR Report (see Results). This was performed to help Herschel.

Results:

qPCR Report (PDF)

qPCR Data File (CFX96)

Initial glance at data looks good. GAPDH exhibits highly consistent Cq values across all samples, controls and exposed. Although, there is slight amplification of something in the two water samples for GAPDH, the melt curve shows that this product has a different melting temperature than our intended target. As such, I believe the GAPDH data to be useable, since no other samples exhibit this smaller product. Defensin shows clean water sample and clean melt curves with a single peak. However, it seems like we may not see an effect on defensin expression in response to the Vibrio vulnificus exposure…

qPCR – C.gigas GAPDH second rep on V.vulnificus exposure cDNA (from 20110311) and standard curves for COX1, COX2, GAPDH

Ran a qPCR on all cDNA samples. Created a standard curve to possibly allow for use of the BioRad software for gene expression analysis. Standard curve was created from pooled cDNA (1uL from each individual sample). Master mix calcs are here.

Results:

qPCR Data File (BioRad CFX96)

qPCR Report (PDF)

Standard curves aren’t that good. Will not use them. Will analyze data using PCR Miner.