20110321

qPCR - C.gigas BB/DH cDNA for PROPS (TIMP3(BB) primers)

Performed qPCR using cDNA from 20110311. This was performed for 2 reps with TIMP3(BB) (SR IDs: 1067 & 1106). Master mix calcs are here. Plate layout, cycling params, etc. can be found in the qPCR Report (see Results).

Results:
qPCR Report (PDF)
qPCR Data File (CFX96)

qPCR - C.gigas BB/DH cDNA for PROPS (HMGP primers)

Performed qPCR using cDNA from 20110311. This was performed for 2 reps with 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:
qPCR Report (PDF)
qPCR Data File (CFX96)



20110315

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)

external image 20110315%20-%20COX%20Tissue%20Distribution%20Gene%20Exp%20Graphs.jpg

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.


20110314

qPCR - C.gigas BB/DH cDNA for PROPS

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:
qPCR Report (PDF)
qPCR Data File (CFX96)

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.



20110311

Reverse Transcription - C.gigas BB/DH DNased RNA (from 20090507) for PROPS

Performed RT on DNased RNA using Promega MMLV RT and Oligo dT according to manufacturer's protocol, using 1ug of DNased RNA, but in a 50uL reaction. Due to large number of samples, cDNA was made in PCR plate. Plate layout and calcs are here.


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.



20110310

SOLiD Sequencing Submission

Submitted the following 8 samples for SOLiD sequencing at HTGU:




20110307

mRNA Isolation - Pooled Black Abalone Dg RNA (from Abalone Dg Exp 1)

mRNA was isolated for SOLiD sequencing by HTGU. Made two pools of San Nick RNA (Control and Exposed) using equal amounts (5ug) of each individual sample. Individual samples used can be found here. mRNA was isolated using Ambion's Micro PolyAPurist Kit according to protocol. Procedure was performed two times on each pool and then EtOH precipitated. Samples were resuspended in 10uL of The RNA Storage Solution provided in the kit and spec'd on the Roberts Lab ND1000. Samples were stored @ -80C in the "Next Gen Sequencing Libraries" box.

Results:

external image 20110307%20mRNA.JPG

Yields are pretty good from both samples (~500ng). However, the OD260/280 values are rather poor.



20110304

3'RACE - C.gigas 3'RACE for COX2

Used Cg_COX2_3'RACE_short (SR ID: 1197) & Cg_COX2_3'RACE_long (SR ID: 1196) and the Clonetech SMART RACE cDNA Amplification Kit (unknown acquisition date) to attempt to acquire more 3' sequence of the C.gigas COX2 isoform. Used Gigas 3'RACE cDNA (from 20080610).

Results:

external image 20110307-01.jpg

Gel Loading:
Lane 1: Hyperladder 1
Lane 2: empty
Lane 3: Cg_COX2_3'RACE_long
Lane 4: Cg_COX2_3'RACE_long NTC
Lane 5: empty
Lane 6: Cg_COX2_3'RACE_short
Lane 7: Cg_COX2_3'RACE_short NTC
Lane 8: Hyperladder 1


No products produced. This could be due to a large number of factors. The age of the cDNA (from 20080610) is well beyond what the Clontech manual says for storage term (6 months). Additionally, the Clontech polymerase used was nearly 6 years old. The kit (and its components) are of an unknown age and could factor in to the failure of this procedure. Also, the primers that were designed had less than ideal Tm, per the kit's recommendations.

May need to sequence some previously purified potential COX2 fragments in order to obtain a more useable region of the gene for RACE.




20110301

NanoDrop1000 Comparison - Roberts vs. Young Lab

A previous comparison was performed (see 20110209), but it was determined that the standard DNA being used to test the machines was old/degraded. Lisa ordered a new standard DNA dilutions series (Quant-iT dsDNA Kit; Invitrogen) and these DNAs were used. All DNAs were measured 5 times and were mixed by gently flicking between each measurement. A "blank" was measured between each different [DNA] and, if the reading was > + or - 1ng/uL, the machine was reblanked.

Results:

Quick assessment is that Graham's NanoDrop1000 is more accurate than ours.

Here is a spreadsheet with averages, standard deviations and experimental error (%). Below are the raw measurements from both machines.

Roberts Lab ND100:

external image 20110301%20Roberts%20Lab%20Quant-iT%20Standards.jpg



Young Lab ND1000:
external image 20110301%20Young%20Lab%20Quant-iT%20Standards.JPG


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 (~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:
qPCR Report (PDF)
qPCR Data File (CFX96)

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.


DNase - C.gigas BB01 (PROPS) RNA (from 20090507

Since the previous DNase treatment failed for this sample, will repeat but will start with less RNA (5ug instead of 10ug). Need more DNased RNA to finish repeating of PROPSSome samples had insufficient quantities of DNased RNA remaining in BB01. Used 5ug of 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): 5ug/1.824ug/uL = 2.74uL RNA + 42.26uL H2O (to 45uL) + 5uL 10X DNase Buffer = 50uL


Results:

external image 20110301%20DNased%20RNA.JPG

RNA looks OK, based on OD260/280. Would like that value to be higher, though. OD260/230 is low, which is typical post-DNased treatment. Will check for residual gDNA via qPCR.



20110228

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:

external image 20110228%20DNased%20RNA-01.JPG

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.


20110225

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:

external image 20110225%20DNased%20RNA.JPG

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.



20110216

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 & 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 PROPSSome 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:

external image 20110217%20DNased%20RNA-01.JPG

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




20110209

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.

external image 20110209%20ABI%207300%20Calibration%20Check%20Amp%20Plot.JPG




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.

external image 20110209%20ABI%207300%20Calibration%20Check%20Melt%20Curves.JPG





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, no 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.

external image 20110209%20ABI%207300%20Calibration%20Check%20Ct%20vs%20Well%20Position.JPG




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.


NanoDrop1000 Comparison - Roberts vs. Young Lab

Due to an apparent reduction in assay sensitivity for the Hematodidium qPCR assay, we have decided to determine if the spec readings of the plasmid DNA being used for the standard curves are accurate. Used C.gigas gDNA and the lambda DNA Standard (100ng/uL) included in the Quant-iT PicoGreen dsDNA Assay Kit (Invitrogen) that was marked as received 9/1/10. Tested both the Roberts Lab and Young Lab using these DNAs. At least 6 separate measurements were taken of each DNA on each machine. Samples were briefly mixed by flicking the tube 4-5 times prior to each measurement.

Results:

Spreadsheet containing absorbance data and calculations of average concentration and standard deviation for both DNA samples on both machines is here.

A quick table of the results:


Roberts Lab
Young Lab
[Avg. gDNA] (ng/uL)
45.656
51.778
Std Dev gDNA
0.2377
0.5825
[Avg. lambda DNA] (ng/uL)
76.01
90.255
Std Dev lambda DNA
3.826
0.9342


The first thing to notice is that the lambda DNA that has been used for standard curves does not have the expected concentration (100ng/uL) on either of the machines. It seems unlikely that BOTH NanoDrop1000s are incorrectly calibrated (which could be a possible explanation for why the lambda DNA is not matching the expected 100ng/uL). This is also supported by recent curves done on the Friedman Lab plate reader using this DNA by Lisa, Vanessa and Elene (data not shown). It's also interesting to note that the lambda DNA also shows a greater standard deviation (on both machines) than the other DNA (gDNA) used in this test. This is surprising as one would expect a store-bought reagent to be of the highest quality, particularly since it is supposed to be used for DNA quantification. However, it should also be remembered that this DNA is over a year old and has never been aliquoted. As such, it has gone through an extremely high number of freeze/thaw cycles which could have an impact on the long-term quality of the DNA.

The second thing to notice is that the Roberts Lab and Young Lab machines provide different concentrations of each of the two DNAs. Unfortunately, due to the fact that the lambda DNA concentration is not as expected (100ng/uL) on either machine it is impossible to determine which machine is more accurate. However, it appears that the Young Lab NanoDrop1000, overall, is more consistently precise in its readings than the Roberts Lab NanoDrop1000. Of course, both machines do seem to be sufficiently precise that precision shouldn't be a concern.

I've notified Lisa of the potentially inaccurate readings of the lambda DNA. She has ordered a fresh set of DNA standards that will be used to test both machines to help assess their accuracy.



20110208

qPCR - Test Young Lab qPCR Calibration (Repeat)

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...



PCR - New C. gigas COX Primers for Sequencing of Isoforms

Used new primers for obtaining bands for additional sequencing of both COX isoforms in C. gigas. Master mix calcs are here. Master mix shorthand (MM##) is described below:


MM07 - Cg_COX_416_F (SR ID: 1193) + Cg_COX1_qPCR_R (SR ID: 1191) Expected band size (if no intron) = ~1540bp

MM08 - Cg_COX_416_F (SR ID: 1193) + Cg_COX2_454align1_R (SR ID: 1190) Expected band size (if no intron) = ~1540bp

MM09 - Cg_COX1/2_qPCR_F (SR ID: 1192) + Cg_COX1_qPCR_R (SR ID: 1191) Expected band size (if no intron) = ~225bp

MM10 - Cg_COX1/2_qPCR_F (SR ID: 1192) + Cg_COX2_454align1_R (SR ID: 1190) Expected band size (if no intron) = ~225bp

MM11 - Cg_COX_1519_F (SR ID: 1146) + Cg_COX2_454align1_R (SR ID: 1190) Expected band size (if no intron) = ~275bp

MM12 - Cg_COX_982_F (SR ID: 1151) + Cg_COX2_454align1_R (SR ID: 1190) Expected band size (if no intron) = ~812bp



Results:

external image 20110208.jpg

Ladder is Hyperladder I from Bioline.
Master mixes are indicated underneath each group by the labels MM##. The order within each MM group (from left to right) is: template, NTC, NTC.
All bands boxed with green were purified using Millipore's Ultrafree-DA spin columns. Samples were stored @ -20C in "Sam's Misc. -20C Box".

MM07 - Fails to produce any bands of any size. Suggests the presence of intron(s) causing the size of the potential amplicon to exceed the capabilities of the polymerase under these cycling conditions.

MM08 - Produces a band of ~400bp which is well below the expected 1540bp (if no introns) size. Due to the faintness of the band, the band was not excised. Will consult with Steven to see if he thinks it worth repeating to produce sufficient product for sequencing.

MM09 - Produce a ~500bp band. The band was excised. This band size is ~275bp larger than the expected size of 225bp. This implies the presence of an intron in this region. This band size differs from that produced by MM10, which suggests that this primer set can be used for qPCR AND distinguish between the COX1 and COX2 isoforms.

MM10 - Produced a ~700bp band. The band was excised. This band size is ~475bp larger than the expected size of 225bp. This implies the presence of an intron in this region. This band size differs from that produced by MM09, which suggests that this primer set can be used for qPCR AND distinguish between the COX1 and COX2 isoforms.


MM11 - Produced multiple bands, of which two were excised; a ~3000bp band and a ~600bp band. These bands were excised solely based on their intensity and their immediate useability for sequencing. Will discuss with Steven on whether or not this should be repeated and the other bands excised for sequencing purposes. Both bands that were excised exceed the expected band size of ~275bp, suggesting the presence of multiple introns. Additionally, the presence of so many products suggests that the primers are not very specific, in regards to their target.

MM12 - An extremely faint band of ~350bp can be seen, however, due to it's faintness and it's small size (expected size was ~812bp), the band was not excised. Will discuss with Steven to see if this warrants repeating to accumulate sufficient product for sequencing purposes. No amplification of any larger products suggests the presence of introns, causing the size of the potential amplicon to exceed the capabilities of the polymerase under these cycling conditions. This is also confirmed by the MM11 PCR results in which a 3000bp band was produced. Since the primer set in MM12 has an additional 600bp at the 5' end, this has already exceeded the abilities of the polymerase, even if this addtional 600bp does NOT include an additional intron. However, it is curious that the MM12 primer set does not produce smaller, spurious PCR products as is seen in the MM11 primer set (these two primer sets both use the same forward primer).





qPCR - Test Young Lab qPCR Calibration (Repeat)

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:

95C - 10min

40 Cycles of:
95C - 15s
55C - 30s
72C - 1m

Melt curve.

Results:

Absolutely no amplification of any kind! Bizarre. Will repeat.



20110204

qPCR - Test Young Lab qPCR Calibration (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:


95C - 10min

40 Cycles of:
95C - 15s
55C - 30s
72C - 1m

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.



qPCR - Test Young Lab qPCR Calibration

Recently, the Young Lab's ABI 7300 qPCR machine was calibrated. Steven asked me to run a plate and see how well the calibration worked. Ran a plate with C.gigas gDNA and Gigas 18s primers (SR ID: 156 and 157) that are known to amplify gDNA. Master mix calcs are here (top half of page). Cycling params were as follows:

95C - 10min

40 Cycles of:
95C - 15s
55C - 30s
72C - 30s

Melt curve.


Results:

Absolutely no amplification of any kind. However, I did use one of our conventional PCR plates and not one of the ABI "prism" plates. Additionally, when I removed the plate from the machine, the plate looked as though it had been vigorously shaken:

external image IMAG0030.jpg

Will repeat this qPCR with a proper ABI "prism" plate.




20110131

Genomic PCR - Repeat of C.gigas COX genomic PCR from 20110118

This was repeated to generate more PCR product for sequencing purposes. PCR master mix calcs and cycling params are here. Master mixes 04 and 05 (MM04 and MM05) were repeated to gain more PCR product from the faint 550bp & 1500bp bands(MM04) and 5000bp band (MM05).


MM04 - Cg_COX_982_F (SR ID: 1151) + Cg_COX_1545_R (SR ID: 1148) Band size w/o intron = ~550bp
MM05 - Cg_COX_982_F (SR ID: 1151) + Cg_COX_2138_R (SR ID: 1149) Band size w/o intron = ~1130bp


Results:

Gel was run on 20110203

external image 20110203%20DNA%20gel.jpg

Samples on the left portion of the gel are from the MM04 primer combo and those on the right are from the MM05. Boxed bands were excised, purified using Millipore Ultra DA-free spin columns and stored @ -20C in Sam's "Misc. -20C Box."

Interestingly in the MM05 set, inconsistent, faint bands of ~400-500bp are visible. These were not visible the first time this PCR was conducted (see 20110118), but the exposure of the gel image wasn't turned up as high as in this image. Due to their inconsistency and extremely low yield, these bands were not excised.



20110128

DNase - DNase C.gigas RNA from 20110120, 20110121 and 20110124

5ug of RNA was DNased using Ambion's Turbo DNA-free kit, following the rigorous protocol (0.5uL of DNase for 30 mins then additional 0.5uL of DNase for 30mins). Calcs for DNase reactions are here. RNA was stored @ -80C in Shellfish RNA Boxes 4 and 5. Samples will be spec'd on Monday.

Results:

external image 20110131%20DNased%20RNA%20ODs.JPG
Overall, the RNA looks really good (based on OD 260/280 numbers). Not surprisingly, the OD 260/230 values for all samples dropped, likely due to the addition of the buffer (salts) used in the DNase reaction. Emma says she will check these samples for residual DNA.

--UPDATE (20110131)-- Emma checked all DNased RNA samples on 20110131 using C.gigas 18s primers (SR ID: ?). She has not listed the results of the whether or not all samples are clean or if some still have residual gDNA carryover.

--UDPATE (20110201-- Samples that appear to have residual gDNA carryover based on Emma's qPCR on 20110131: Muscle C6, Gill 1hr C2 & E2.




20110124

RNA Isolation - Various C.gigas Tissue from 20110111

RNA was isolated in 1mL TriReagent, according to protocol. Samples were resuspended in 50uL 0.1% DEPC-H2O and spec'd. RNA was stored @ -80C in "Shellfish RNA Box #4

Results:

external image 20110124%20RNA.JPG


RNA looks OK. Not surprising, but mantle and Dg/Gonad tissues ended up with poor OD260/230 ratios. This has been observed in the past with these tissue types.





20110121

RNA Isolation - Various C.gigas Tissue from 20110111

RNA was isolated in 1mL TriReagent, according to protocol. Samples were resuspended in 50uL 0.1% DEPC-H2O and spec'd. RNA was stored @ -80C in "Shellfish RNA Box #4".

Results:


external image 20110121%20RNA-01.JPG


Overall, all RNA looks very good (based on 260/280 and 260/230 values).




20110120

RNA Isolation - Various C.gigas Tissue from 20110111

RNA was isolated in 1mL TriReagent, according to protocol. Samples were resuspended in 50uL 0.1% DEPC-H2O and spec'd. RNA was stored @ -80C in "Shellfish RNA Box #4".

Results:

external image 20110120%20RNA-01.JPG

All gill RNA looks nearly perfect (based on 260/280 and 260/230 values). Muscle RNA is only OK (based on 260/280 and 260/230 values).



20100118

Genomic PCR - C.gigas cyclooxygenase (COX) genomic sequence

Attempt to obtain full genomic sequence for C.gigas COX. PCR set up/cycling params/etc are here. Primer set combinations(master mixes) are as follows:

MM01 - Cg_COX_5'UTR_3_F (SR ID: 1150) + Cg_COX_1009_R (SR ID: 1147) Band size w/o intron = ~1000bp
MM02 - "" + Cg_COX_1545_R (SR ID: 1148) Band size w/o intron = ~1540bp
MM03 - "" + Cg_COX_2138_R (SR ID: 1149) Band size w/o intron = ~2135bp

MM04 - Cg_COX_982_F (SR ID: 1151) + Cg_COX_1545_R (SR ID: 1148) Band size w/o intron = ~550bp
MM05 - "" + Cg_COX_2138_R (SR ID: 1149) Band size w/o intron = ~1130bp

MM06 - Cg_COX_1519_F (SR ID: 1146) + Cg_COX_2138_R (SR ID: 1149) Band size w/o intron = ~620bp


Results:

external image 20110118.jpg

Bioline Hyperladder I used for marker. Gel is loaded with template samples at the far left of each master mix group with two no template controls (NTC) in the remaining two wells of each master mix group. All NTCs on the gel are clean.

All bands surrounded by a green box were excised from the gel.

MM01, MM02 and MM03 - The smallest expected band (i.e. no intron present) would have been 1000bp in MM01. Instead, we see faint banding of multiple sizes less than 1000bp in both MM01 and MM02. MM03 fails to produce any bands. This potentially suggests a couple of things. Firstly, the multiple banding produced in MM01 and MM02 suggests that the PCR conditions lead to some non-specific priming and should be optimized. Secondly, the fact that no bands were produced that are equal to or larger than the "no intron size" suggests that intron(s) may exist in the 5' region of the COX gene and are large enough that the polymerase had insufficient time/ability to amplify.

MM04 - Three distinct bands were produced: 2000bp, 1500bp and 550bp. The size of band that would have been produced had an intron NOT been present would have been ~550bp. A band of this size was produced in this PCR reaction. However, two additional bands were produced. The presence of these two larger bands lends additional evidence for the existence of multiple isoforms of COX (which is also supported by the fact that multiple isoforms of COX are known to exist in most other species). The 2000bp band was excised and purified with Millipore Ultra-free DA spin columns and stored @ -20C until a sequencing plate is readied.

MM05 - A distinct band of ~5000bp was produced. This is significantly larger than the "no intron size" of ~1130bp, suggesting the presence of an intron. This band was excised, but not purified due to the low concentration of DNA in the gel. The gel slice was stored @ -20C until this PCR reaction could be repeated to accumulate sufficient product for sequencing.

MM06 - A distinct band of ~2200bp was produced. This is significantly larger than the "no intron size" of ~620bp, suggesting the presence of an intron. The band was excised and purified with Millipore Ultra-free DA spin columns and stored @ -20C until a sequencing plate is readied.


The PCR reactions reveal the presence of intron(s) in the COX gene we're investigating as well as providing evidence for the existence of multiple isoforms in C.gigas. Since the PCR products that have been excised for sequencing are so large, additional primers will need to be designed closer to the introns in order to generate smaller products that can be fully sequenced. Additionally, all reactions using the primer designed to anneal in the 5'UTR of COX (SR ID: 1150) failed to produce useful results. This is either due to poor performance of the primer under these reaction conditions or due to the presence of a large intron in the 5' region of the gene. Additional reverse primers will be designed that anneal closer to the 5' portion of the COX gene in hopes of characterizing the 5' genomic sequence better.

After speaking with Steven today about the potential existence/"discovery" of multiple isoforms, he decided to map the newly-released C.gigas 454 NGS data to the existing COX coding sequence in GenBank (FJ375303). The alignment is shown below.

external image 20110119%20CLC%20Genomics%20Gigas%20COX%20454%20Alignment.jpg

The two 454 reads that map closest to the 5' end of the COX coding sequence match up nearly perfectly, with periodic SNPs. The remaining 454 reads that map to the COX coding sequence are very different and provide very good evidence of a previously unidentified isoform of COX in C.gigas. Primers will be designed from both the existing COX sequence in GenBank (FJ375303) and the other potential isoform. These primers will likely be used in both qPCR and for sequencing purposes, in order to be able to distinguish and characterize both isoforms. Additionally, BLASTing will be performed with the sequences from both isoforms to evaluate how they match up with existing COX isoforms in other species.




20110111

Bacterial Dilutions - Determination of Colony Forming Units from Gigas Bacterial challenge (from earlier today)

All dilutions were performed with 1x LB+ 1%NaCl. 100uL were plated of all dilutions (see below) on 1xLB+1%NaCL plates. Plates were incubated O/N @ 37C. Colonies will be counted tomorrow to determine CFU for each sample.

Plated 100uL of:

V.vulnificus, t=0, 1:1,000,000 and 1:10,000,000
V.vulnificus H2O sample, t=1 & 3, 1:10,000 and 1:1,000,000
V.tubiashii, t=0, 1:1,000,000
Control H2O sample, t=1 & 3, Undiluted

Samples tubes containing bacteria and dilutions were stored @ 4C.


*UPDATE 20110112*:

Colony counts and calculations

V.vulnificus 0hr = Both dilutions produced a total lawn of bacteria. Uncountable. Will plate higher dilutions, but this will now only be a rough estimate due to the time that has passed.

---UPDATE---
New serial dilutions (90uL plated) of V.vulnificus 0hr were performed, down to 10^12. The only countable plate was the 10^12 dilution.

V.vulnificus 0hr = 10^12 dilution x 410 CFU = 4.1x10^14 CFU/90uL = 4.56x10^12 CFU/uL x 8x10^6uL (8L H2O in oyster tank) = 3.64x10^19 CFU total in oyster tank/8L = 4.56x10^18 CFU/L

V.vulnificus 1hr = 1:1,000,000 dilution x 48 CFU = 4.8x10^7 CFU/100uL = 4.8x10^5 CFU/uL x 8x10^6uL (8L H2O in oyster tank) = 3.84x10^12 CFU total in oyster tank/8L = 4.8x10^11 CFU/L
V.vulnificus 3hr = 1:1,000,000 dilution x 23 CFU = 2.3x10^7 CFU/100uL = 2.3x10^5 CFU/uL x 8x10^6uL (8L H2O in oyster tank) = 1.84x10^12 CFU total in oyster tank/8L = 2.3x10^11 CFU/L

Control H2O (no significant growth occurred O/N, just tiny colonies; continued incubation to allow colonies to increase in size for easier counting)

Control H2O 1hr = Undiluted 146 CFU/100uL = 1.46 CFU/uL x 8x10^6uL (8L H2O in oyster tank) = 1.168x10^7 CFU total in oyster tank/8L = 1.46x10^6 CFU/L
Control H2O 3hr = Undiluted 106 CFU/100uL = 1.06 CFU/uL x 8x10^6uL (8L H2O in oyster tank) = 8.48x10^6 CFU total in oyster tank/8L = 1.06x10^5 CFU/L

V.tubiashii 0hr = 1:1,000,000 dilution x 31 CFU = 3.1x10^7 CFU/100uL = 3.1x10^5 CFU/uL x 5x10^4 (50mL total volume of bacteria) = 1.55x10^10 CFU total V.tubiashii



Gigas Bacterial Challenge - 1hr & 3hr Challenges with Vibrio vulnificus

400mL O/N culture (1x LB+1% NaCl, 37C, 150RPM, 1L flask) of V.vulnificus (STRAIN??) and V.tubiashii (Strain: RE22) were pelleted (4300RPM, 25C, Sorvall ST-H750 Rotor). Supe was removed and pellets were each resuspended in 50mL sea water. 1mL was taken from each to use for dilutions to determine colony forming units (CFU).

Two containers were set up with each containing 16 C.gigas, and air stone and 8L of sea water. The entire 50mL of V.vulnificus was added to one of the containers. 8 oysters were sampled (gill and mantle tissue) from each container at 1hr and 3hrs after the addition of V.vulnificus culture and immediately frozen on dry ice. Samples were stored @ -80C in the "Gigas Vibrio Exposure 1,3hrs 1/11/11" box. Additionally, 1mL samples of the water were taken at each time to determine CFU in the water.

In addition to the samples taken above, the following tissues were taken from 5 control oysters at the 3hr time point and treated/stored in the same fashion as the others, specifically for assessment of cyclooxygenase tissue distribution analysis: muscle, digestive gland/gonad (difficult to differentiate)

All oysters were measured. Morphometric data is here.