Tag Archives: RNA isolation

RNA Isolation – Colleens’ Sea Star Coelomycetes Samples

Isolated RNA from the following samples stored in RNAlater:

  • TH52 3.28.14 c-fluid
  • TH54 3.28.14 c-fluid
  • CH55 3.28.14 c-fluid
  • CH56 3.28.14 c-fluid
  • CH57 3.28.14 c-fluid
  • TH65 3.28.14 c-fluid
  • TH66 3.28.14 c-fluid
  • TH67 3.28.14 c-fluid

Spun samples 5000g, 20mins @ RT to pellet any cells. Discarded supe. Resuspended cells/debris in 1mL TriReagent. Disrupted cells by pipetting and vortexting. RNA was isolated using the Direct-zol RNA Miniprep Kit (ZymoResearch). RNA was DNase treated on-column, as described in the manufacturer’s protocol, using DNase I. RNA was eluted from the columns using 25uL of nuclease-free H2O and spec’d on a NanoDrop1000.

Results:

So, this is disheartening. Overall, the RNA looks pretty crappy; poor 260/280 ratios and a general shift in absorbance to 270nm. Plus, the yields aren’t that great. Maybe RNA left on the column and/or some sort of contaminant pushing these readings out of whack?

I will perform another elution on the columns with 50uL of nuclease-free H2O and spec that elution set:

There’s still a shift in the peak absorbance in most samples to 270nm… I’m going to combine the two sets of elutions and spec:

Although the 260/280 values are significantly better, there’s still this persistent shift of peak absorbance to 270nm. I contacted technical support for the kit and they say the absorbance shift is indicative of phenol contamination. They have advised that I add a volume of TriReagent to the RNA and re-run it through a new set of columns, following the entire RNA isolation protocol.

RNA Isolation – Sea Star Coelomocytes (from Colleen)

Isolated RNA from two samples stored in RNAlater that had either no visible pellet or a minutely visible pellet:

  • Control P26
  • Filt. Inj. P8

Samples were spun 5000g, 20mins @ RT. Supe was removed, being sure to leave behind any debris that failed to pellet. Samples were homogenized in 1mL of TriReagent by pipetting/vortexing. RNA was then isolated using the Direct-zol RNA Miniprep Kit (ZymoResearch). RNA was eluted from the column with 25uL of 0.1%DEPC-treated H2O and spec’d on a NanoDrop1000.

Results:

RNA quality looks very good, as do the yields. I’m very surprised I got anything close to 1ug out of either sample!

However, it should be noted that neither of these samples has been DNased and, as such, the yields seen above may potentially include residual gDNA carryover which would artificially inflate the yields seen above. Will DNase the samples to see how yields are affected (if at all).

RNA Isolation – Sea Star Coelomocytes (provided by Colleen Burge)

Tried another method of RNA Isolation for comparison with regular TriReagent method.

Used the Direct-zol RNA MiniPrep Kit (Zymo Research) on the following samples stored in RNAlater:

  • P6 Control
  • P16 Filt. Inj.

Pelleted samples in RNAlater by spinning 5000g, 10mins @ RT. Removed RNAlater, lysed pellets in 1mL TriReagent. Split each sample equally into two tubes (500uL in each tube). Added equal volumes of 100% ethanol to each tube and vortexed. Transferred samples to spin columns and followe manufacturer’s protocol. Eluted with 25uL of nuclease-free H2O (provided in kit). Spec’d on NanoDrop1000.

Results:

RNA quality is very good (based on 260/280 ratios). This turned out much better than the previous attmpt using the basic TriReagent method. However, the previous attempt (see 20140401) may have been compromised by me being too aggressive when collecting the aqueous phase. Knowing how little sample was present, I may have been overzealous in trying to gather too much of the aqueous phase, leading to the phenol carryover that was evident.

Regardless, these columns seem to do an excellent job of eliminating even salt carryover, as we frequently see high absorbance at 230nm with marine samples; particularly those stored in RNAlater.

RNA Isolation – Sea Star Coelomocytes (provided by Colleen Burge)

Isolated RNA from the following samples (stored in RNAlater):

P18 Control 3/17/14

P10 Filt. Inj. 3/17/14

These were “trial” RNA isolation runs to determine what yields we could expect from samples of this nature.

Both samples had very small tissue/cell pellets. Tubes were spun @ 5000g for 10mins at RT to ensure all cells were pelleted. RNAlater was removed and pellets were lysed using 1000uL of TriReagent, supplemented with 8uL of PolyAcryl carrier. PolyAcryl Carrier was used to enhance RNA recovery from such small starting materials. Remainder of procedure followed manufacturer’s protocol. RNA was resuspended in 20uL of 0.1% DEPC-H2O and spec’d on a NandoDrop1000.

Results:

As can be seen by the absorbance spectrum plots (top image), there is clear phenol contamination (indicated by shift of absorbance peak to 270nm, instead of the peak being at 260nm). Additionally, there’re large peaks at 230nm in each of the two samples, suggesting other contamination (high residual salts, ethanol?). Additionally, the 260/280 ratios are subpar for RNA quality (i.e. <1.9). However, these ratios could be skewed by the the residual phenol present in both samples. I may perform an ethanol precipitation on these just to see if I can get them cleaned up.

Yields for both samples are very promising.

RNA Isolation – C.gigas Larvae from Taylor Summer 2011

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.

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.)

 

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 (#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.

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.

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: