Tag Archives: Vibrio exposure

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…

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qPCR – C.gigas COX1/COX2 Tissue Distribution

Performed qPCR using pooled cDNA from 20110311. Pooled 2uL from each of the following samples groups: Dg 3hr C, Gill 1hr C, Gill 1hr E, Mantle 3hr C, and Muscle 3hr C. Master mix calcs are here. Plate layout, cycling params, etc can be found in the qPCR Report (see Results). Primers sets run were:

EF1_qPCR_5′,3′ (SR IDs: 309, 310)

Cg_COX1/2_qPCR_F (SR ID: 1192) + Cg_COX1_qPCR_R (SR ID: 1191)- Target = COX1

Cg_COX1/2_qPCR_F (SR ID: 1192) + Cg_COX2_454align1_R (SR ID: 1190) – Target = COX2

Results:

qPCR Report (PDF)

qPCR Data File (CFX96)

Graphs were generated using the BioRad CFX Manager v2.0 software. Expression was normalized to EF1. Also to note, gene efficiency was assumed as 100% by the software since no standard curve was run on the plate. As such, analysis of this data may not be exact.

It’s clear by examining the graphs that the primers being used to differentiate COX1 and COX2 (since they share a common primer: SRID 1192) are differentially expressed. This indicates that the primer sets are indeed amplifying different targets as hoped. This was the primary intention of this qPCR. However, we also now have an idea of tissue distribution of the two genes, as well as their response to V. vulnificus exposre after 1hr. Next step is to perform this qPCR on all the individuals from this experiment as well as the different tissues.

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Reverse Transcription – C.gigas DNased RNA (from 20110131) from V.vulnificus Exposure & Tissues (from 20110111)

Performed RT on DNased RNA using Promega MMLV RT and Oligo dT according to manufacturer’s protocol, using 1ug of DNased RNA. Due to large number of samples, cDNA was made in PCR plate. Plate layout and calcs are here. cDNA was diluted 4-fold (to 100uL total volume) based on qPCR done by Emma on 20110202.

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qPCR – COX qPCR Vibrio Exposure Response Check

Used COX primers (SR IDs 1060, 1061) and cDNA from 20080327, which consisted of 7 control gigas gill and 7 vibrio-exposed (24hrs) gigas gill samples, labeled as C# and VE#, respectively. The experiment was a 24hr. exposure live Vibrio vulnificus, parahaemolyticus Cf = 2.055×10^11 (6.85×10^7 Vibrio cells/oyster).
Note: Used a free sample of 2x Brilliant III Ultra Fast SYBR Green QPCR Master Mix (Stratagene) for this qPCR. Mixed components and set up cycling params according to the manufacturer’s recommendation for the BioRad CFX96.

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

Results:

qPCR Report (PDF).

PCR Miner analysis is here. There appears to be an increase in COX expression in samples exposed to Vibrio sp. (see graph below), however, I have not determined if the results are significant.

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gDNA Isolation – Various gigas samples (continued from yesterday)

Pelleted residual tissue 10mins @ 10,000g @ RT. Transferred supe to new tubes. Precipitated DNA with 0.25mL 100% EtOH. Incubated 3mins @ RT. DNA was pelleted 5mins @ 5000g @ RT. Supe was removed, pellets were washed with 1mL 75% EtOH (x2). Supe was fully removed and the DNAs were resuspended in 300uL 8mM NaOH (made 7/9/10 SJW).

1M HEPES (provided with DNAzol) was added at a 1:100 dilution to achieve a pH = 8.0. This was based on the DNAzol protocol calculations (For 1mL of 8mM NaOH, use 101uL of 0.1M HEPES = pH 8.0).

Samples were spec’d on NanoDrop 1000. Used a sample with 8mM NaOH and 1M HEPES as a blank to match the pH = 8.0 of the samples.

Results:

260/280 ratios look good for all samples. Most of the samples have mediocre 260/230 ratios. Yields are excellent for all samples.

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gDNA Isolation – Various gigas samples

Placed ~20mg fragments of tissue in 250uL DNAzol. Added 1.35uL of Proteinase K (Fermentas; 18.5mg/mL) to reach a final concentration of 100ug/mL. Incubated RT, O/N, end-over-end rotation. Will complete DNA isolation tomorrow.

Sample List:

Vt Gigas Live #3 Gill 24E (from 20080828; Tatyana’s notebook)

Gigas Control #2 Gill 24E (from 20080828; Tatyana’s notebook)

NB-1209-10 (RNA Later)

SB-1209-14 (RNA Later)

WB-1209-09 (RNA Later)

0629 gill 5aza

0629 gonad 5aza

0629 mantle 5aza

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Vibrio challenge CONTINUED (from yesterday)

Significantly more bacteria in the container containing autoclaved oysters. Collected 2 x 50mL from each treatment. Collected ~750mL from each treatment. Cells were pelleted 4000RPM, 15mins, 4C. Supe was removed and pellets frozen @ -80C.

100uL of a 1:1,000,000 dilution of the control bacteria were plated on 1x LB + 1% NaCl plates and incubated O/N @ RT. 100uL of a 1:10,000,000 dilution of the exposed bacteria were plated on 1x LB + 1% NaCl plates and incubated O/N @ RT. Control colony count the next day = 563 colony forming units (CFU). Exposed colony count the next day = 367 CFU.

Control Calculations
563 CFU/100uL = 5.63 CFU/uL
5.63 CFU/uL x 1:1,000,000 dilution = 5.63 x 10^6 CFU/uL
5.63 x 10^6 CFU/uL x 1000uL/mL = 5.63 x 10^9 CFU/mL

Exposed Calculations
367 CFU/100uL = 3.67 CFU/uL
3.67 CFU/uL x 1:10,000,000 dilution = 3.67 x 10^7 CFU/uL
3.67 x 10^7 CFU/uL x 1000uL/mL = 3.67 x 10^10 CFU/mL

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Vibrio challenge CONTINUED (from yesterday)

500mL culture was split evenly between two containers containing 3L sterile sea water each. One container also contained 3 large, autoclaved C. gigas. Containers had an air stone to promote circulation. 4 x 1mL samples were collected from each container, pelleted @ 10,00RPM 1min. Supe removed and samples stored @ -80C. Samples will be collected @ t = 0, 0.5, 1.0 and 24 hrs. Containers were covered with aluminum foil to minimize splashing caused the by the air stone.

100uL of a 1:1,000,000 were plated on 1x LB + 1% NaCl plates and incubated O/N @ 37C. Colony count the next day = 399 colony forming units (CFU).

399 CFU/100uL = 3.99 CFU/uL
3.99 CFU/uL X 1:1,000,000 dilution = 3.99 x 10^6 CFU/uL
3.99 x 10^6 CFU/uL x 1000uL/mL = 3.99 x 10^9 CFU/mL
3.99 x 10^9 CFU/mL x 250 mL/container = 9.975 x 10^11 CFU/container

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