Tag Archives: TriReagent

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.

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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.

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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.

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RNA Isolation – Hard Clam Gill Tissue from Vibrio Experiment (see Dave’s Notebook 5/2/2011)

Isolated RNA in 1mL of Tri-Reagent according to the manufacturer’s protocol. Also, finished RNA isolation of samples that were started 20110506. Samples were resuspended in 50uL 0.1%DEPC-H2O and spec’d.

Results:

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RNA Isolation – Hard Clam Gill Tissue from Vibrio Experiment (see Dave’s Notebook 5/2/2011)

Isolated RNA in 1mL of Tri-Reagent according to manufacturer’s protocol. Samples were precipitated with isopropanol and stored over the weekend @ -20C. Will conclude isolation on Monday. The samples isolated were:

MA 1-11

MA Vt 1-11

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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:

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.

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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:

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

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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:

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).

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RNA Isolation – Hard Clam Tissues Rec’d from Rutgers on 20100820

RNA was isolated from the following samples using TriReagent, according to protocol:

MAX 1, 2, & 3

CA 1, 2, & 3

MA 1, 2, & 3

Samples were resuspended in 50uL of 0.1% DEPC-H2O and spec’d:

Results:

260/280 ratios are decent for most of the samples, with MAX1 and MAX 2 being the exception. Both of these samples also have very poor 260/230 ratios. Out of curiosity, I will EtOH precipitate all samples to see if I can improve both the 260/280 and 260/230 ratios.

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RNA Isolation – Sepia samples

Isolated RNA from 7 sepia samples received 20091125. Samples were removed from RNA Later, blotted and homogenized in 500uL of TriReagent. 500uL of additional TriReagent was added to the tubes after homogenization. Procedure was followed normally. The sepia retina RNA was isolated separately from the other samples and was resuspended in 100uL of 0.1% DEPC-H2O. The remaining samples were isolated and resuspended in 20uL of 0.1% DEPC-H2O. Nearly all samples had some sort of purple tint to them, ranging from almost black to extremely faint purple hue. The samples were spec’d and then stored @ -80C in Sam’s RNA Box #1.

Results:

Nearly all of the samples exhibited very strange curves and mediocre 260/280 ratios (for RNA). Could be due to the “purple stuff” carryover or possibly an effect of the RNA Later from which the samples were stored.

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