Treated 5ug of total RNA (in a 50uL rxn) using Turbo DNA-free (Ambion) according to the “Standard” protocol. Samples were spec’d on the Roberts Lab NanoDrop 1000.
Results:
All samples look good, both quality and quantity-wise. Will check for residual gDNA in these samples via qPCR.
Samples that had been split from earlier today (see the RNA Isolation below) were resuspended in 1mL of DNAzol (MRC). 100ug of Proteinase K (Fermentas) was added to each sample. Samples were incubated at RT, O/N on a rotator. On 20120427 samples were pelleted 10mins, 10,000g, and supe transferred to fresh tube. DNA was precipitated with 0.5mL of 100% EtOH, mixed gently and pelleted 5mins, 5000g. Supe was discarded, pellets were washed with 1mL 75% EtOH, re-pelleted at same speed as previous step, supe discarded and pellets were resuspended in NanoPure H2O. Samples were spec’d on the Roberts Lab NanoDrop1000.
Results:
Report on the NanoDrop software wouldn’t display, so I’ve entered the concentration of each sample in the table below.
| SampleID | ng/uL |
| 201 | 1364 |
| 280 | 131.7 |
| 314 | 710.3 |
| 342 | 539.4 |
| 434 | 274.4 |
| 552 | 334.8 |
| 605 | 369.7 |
Samples had been stored in RNA Later (Ambion). Samples were pelleted and the RNA Later supe removed. Samples were washed (2x) with 1mL TE (pH = 8.0) to remove excess salt resulting from the RNA Later. Samples were split, roughly equally, into two separate tubes. Samples were pelleted and the supe removed. One tube from each sample was set aside for gDNA isolation using DNAzol (MRC). The other tube was vortexed vigorously in TriReagent (MRC) and the then treated according to protocol. Samples were resuspended in 100uL of 0.1% DEPC-H2O and spec’d on the Roberts Lab NanoDrop 1000.
Results:
Overall, the samples look really good. Some samples (280, 434 & 605) required re-specing after the NanoDrop was reblanked in order to get a reading without an error message. They will be DNased and then reverse transcribed.
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).
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.
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.
Extracted DNA from the following water filter samples using the Qiagen DNeasy Blood & Tissue Kit:
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.
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 M-MLV RT protocol). Samples were prepared in a plate and stored @ -20C. Plate layout and all reverse transcription calcs are here:
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.
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.)
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:
