Tag Archives: Vibrio tubiashii

qPCR – Detection of V.tubiashii Presence and Expression Using VtpA Primers in DNA/cDNA from yesterday

Ran qPCR with VtpA primers on cDNA and DNA (from yesterday) of C.gigas larvae to see levels of V.tubiashii compared to their water filter samples (see 20120326). Master mix calcs are here. Plate layout, cycling params, etc can be seen in the qPCR Report (see Results). Used 1uL of cDNA and 100ng (1uL) of DNA as template.

All samples were run in duplicate.

Results:

qPCR Data File (CFX96)
qPCR Report (PDF)

No detectable levels of expression (or, no expression at all) in any of the cDNA samples.

Below I’ve put together a very rough comparison of larvae levels, based off of the the standard curve. I have NOT done the full back calculations!! This is data straight out of the qPCR machine, using the standard curve. Due to the large range, I’ve graphed the data on a logarithmic scale so all the data is visible on the graph.

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Reverse Transcription – DNased C.gigas Larval RNA from 20120427

Performed reverse transcription using random primers (Promega) diluted 1:100 (5ng/uL) with 175ng of DNased total RNA. Random primers were used because we will be targeting V.tubiashii RNA instead of eukaryotic RNA. Reverse transcription was performed with M-MLV Reverse Transcriptase (Promega) according to the manufacturer’s protocol. Calcs are here.

 

Created dilutions of all samples to 100ng/uL in a volume of 50uL in preparation for qPCR analysis. Calcs are here.

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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.

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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).

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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.

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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.

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DNA Extraction – Taylor Water Filter Samples from 2011

Extracted DNA from the following water filter samples using the Qiagen DNeasy Blood & Tissue Kit:

  • 158
  • 200
  • 279
  • 313
  • 341
  • 410
  • 433
  • 503
  • 551
  • 604

Filters were cut into ~13 pieces and placed in 1.5mL snap cap tubes containing 50uL of Proteinase K and 400uL of Buffer AL. Samples were incubated O/N @ 56C. Tubes were spun @ 16,000g @ RT for 2mins. 400uL of 100% EtOH was added to each tube and vortexed. Tubes were spun @ 16,000g @ RT for 2mins. Supe was transferred to Qiagen column. Qiagen protocol was followed from this point on. Samples were eluted with 100uL of Buffer AE and stored @ 4C.

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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).

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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.

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