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.

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.

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).

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.

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.

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.

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 not true for all of the samples.