20120326
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).
20120323
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 (CFX)
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 (CFX)
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.
20120322
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.
20120312
Reverse Transcription - Dave's Manila Clam (Venerupis philippinarum) DNased RNA from 20120307 and 20120302
Performed reverse transcription on 1.5ug of DNased RNA in a 75uL reaction, using oligo dT primers. All reagents were scaled appropriately (based on Promega's MMLV RT protocol). Samples were prepared in a plate and stored @ -20C. Plate layout and all reverse transcription calcs are here.
20120308
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.
20120307
DNase - Dave's Manila Clam (Venerupis philippinarum) RNA from earlier today
DNased RNA using Ambion's Turbo DNA-free Kit following the "routine" protocol. 5ug of total RNA from each sample was treated in 50uL reactions. Samples will be spec'd on Monday with the Roberts Lab NanoDrop 1000.
Results:
RNA Isolation - Dave's Manila Clam (Venerupis philippinarum) Gill Samples (#25-48)
Isolated RNA from Manila Clam gill samples provided by Dave, according to protocol. Samples were resuspended in 0.1%-DEPC H2O and spec'd on the Roberts Lab NanoDrop1000. Samples were stored @ -80C in Dave's box that the tissue was initially stored in.
Results:
All samples look great with excellent yields and great 260/280 values. Will proceed with DNasing. (Note: Sample #42 appears twice because the first reading had an air bubble and, as such, should be discarded.)
20120305
Sequence Manipulation - Emma's Translated C. gigas Sigenae v8 FASTA File
The UW proteomics facility requested a protein FASTA file without stop codons (marked with asterisks in the FASTA file) in the middle of the amino sequences provided. To accomplish this, I first created a new history in
Galaxy. Then, I uploaded the C. gigas Sigenae v8 Transcriptome Contigs FASTA file (I had previously downloaded this) to
Galaxy. I then used the "getorf" app in the EMBOSS package to translate all regions between start and stop codons. The file output was a FASTA file and made public. The
link to this data file was sent to Philip at the UW proteomics facility for download and subsequent analysis.
(Note: Link to the data file above is active as of 20120305. This may not be permanent due to a number of factors, one of which being the simplicity with which this data can be regenerated.)
20120302
DNase - Dave's Manila Clam (Venerupis philippinarum) Gill RNA from Yesterday
DNased RNA using Ambion's Turbo DNA-free Kit following the "routine" protocol. 5ug of total RNA from each sample was treated in 50uL reactions. Samples will be spec'd on Monday with the Roberts Lab NanoDrop 1000.
Results:
20120301
RNA Isolation - Dave's Manila Clam (Venerupis philippinarum) Gill Samples (#1-24)
Isolated RNA from Manila Clam gill samples provided by Dave according to protocol. Samples were resuspended in 0.1%-DEPC H2O and spec'd on the Roberts Lab NanoDrop1000. Samples were stored @ -80C in Dave's box that the tissue was initially stored in.
Results:
Overall, RNA quality is very good, as well as yields.
20120209
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).
20120208
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.
Reverse Transcription - C.gigas larvae DNased RNA (from 20120125)
Performed reverse transcription on Dnased RNA from 20120125 using 175ng RNA from each sample. Also used random primers (instead of the usual Oligo dT primers) since these samples will also be used to analyze gene expression in Vibrio tubiashii.
cDNA calcs are here.
20120125
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.
DNAse - C.gigas RNA from 20120124
5ug of each RNA was DNased using Ambion's Turbo DNA-free Kit, according to the rigorous protocol and spec'd on Roberts Lab NanoDrop 1000.
RNA volume calcs are here.
Results:
DNased RNA looks fine. Low OD260/280 ratios, but this is often seen after DNase treatment and particularly with low [RNA]. Will perform qPCR to assess gDNA removal.
20120124
RNA Isolation - C.gigas Larvae from 20110412 & 20110705 (Continued from 20120112)
All of the RNA samples were re-combined with their respective counterparts and subject to a standard EtOH precipitation (0.1 volumes of 3M NaOAc, pH = 5.2, 2.5 volumes 100% EtOH; incubated -80C 1hr; pelleted; washed with 1mL 70% EtOH; pelleted). Pellets were washed two additional times (for a total of three washes) with 70% EtOH. RNA was resuspended in 50uL of 0.1% DEPC-H2O and spec'd on the Roberts Lab NanoDrop 1000.
Results:
Yields for the 4/12/2011 samples were all lower than the yields for the 7/5//2011 samples. However, the RNA quality (based on OD260/280 ratios) looks pretty good for both groups of RNA. RNA will be treated with DNase before reverse transcription.
20120112
RNA Isolation - C.gigas Larvae from 20110412 & 20110705
RNA was isolated from C.gigas larvae collected from Taylor Shellfish hatchery on the dates noted above. Samples were in RNA Later. RNA Later was removed. Attempted homogenization with a pestle proved futile, as a significant quantity of larvae were sticking to the pestle and were nearly impossible to wash off using TriReagent as a rinsing agent. Due to this, all samples were vortexed for 1min in 1mL of TriReagent. It should be noted that the TriReagent took on a cloudy appearance and even showed some separation into two layers upon letting the samples sit. This was not normal and I was immediately concerned about the high salt content from residual RNA Later. Samples were treated normally with the following changes:
- Aqueous phase after chloroform treatment was clear, but grey in color. This is not necessarily unusual.
- Addition of isopropanol triggered immediate precipitation of a dark grey material.
- "Pelleting" of the RNA after the isopropanol precipitation resulted in a gooey grey material that did NOT pellet, and a clear supernatant. The grey goo was transferred to a clean tube. An additional 500uL of isopropanol was added to the clear supernatant of two samples (#140 & #142), as well as to the grey goo. The addition of isopropanol to the clear supe resulted in an immediate precipitation of white salt-like material. The isopropanol appeared to have no effect on the grey goo. All samples were stored @-20C in their existing conditions until 20120116.
- Since the two samples that were treated with an additional 500uL of isopropanol produced an excess of salt precipitation, I instead added 1mL of 70% EtOH to all the remaining samples; both the clear supernatants and the grey goo. The idea being that the higher water content in the 70% EtOH would help to keep the salts in solution, while precipitating the RNA. Samples were pelleted. All of the grey goo samples produced a white pellet. The grey goo seemed unchanged. Supernatants (including grey goos) were discarded and the resulting pellets from all samples were washed in this fashion were washed three more times.
- Pellets were resuspended in 25uL of 0.1% DEPC-H2O and stored @ -80C until 20120123.
- Samples were spec'd on the Roberts' Lab NanoDrop 1000.
Results:
Spreadsheet of OD readings is here.
Since samples were split into two (clear supernatant and grey goo), they were kept separate through the remainder of the process. Sample names are appended with "-1" or "-2". "-1" samples are grey goo samples and the "-2" samples are the clear supernatant samples.
Overall, most of the grey goo samples appear to have produced the highest yields and highest quality of RNA, although this is no true for all of the samples.