Tag Archives: RNA quantification

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.

Chloroform Clean Up – Lexie’s QPX RNA from 20110504

After submission of QPX samples to HTGU for Illumina library prep yesterday, I was notified that there was insufficient RNA for the QPX RNA samples. I checked the source RNA on the Roberts Lab NanoDrop1000 and determined that they had high phenol contamination (large peak at 270nm), which results in a large exaggeration in the OD260 absorbance (NanoDrop1000 report[JPEG]; notice terrible OD260/280 ratios; did not save screen shot of absorbance peaks.). As such, the concentrations that Lexie had listed in her notebook for these samples are highly inaccurate and highly inflated. To remove the phenol, I brought all of her QPX RNA samples from 20110504 up to ~200uL with 0.1%DEPC-H2O, added 200uL of chloroform, vortexed for 30s, spun at 12,500g RT for 15mins, and transferred aqueous phase to new tube. Then performed an ethanol precipitation on the aqueous phase. Added 0.1 vols of 3.0M sodium acetate (pH = 5.2), 2.5 vols of 100% EtOH, mixed and incubated at -20C for 1hr. Pelleted RNA by spinning at 16,000g 4C for 15mins.

Results:

As suspected, most of these samples have absolutely no RNA in them. However, the samples that do (the “Control” samples), look great! Pooled 2ug each of the RT Control a & b samples and pooled 2ug each of the 10C Control a & b samples (which are ATCC). Calculations are here. Will take them down to HTGU tomorrow to replace the bad samples that were provided yesterday.

DNase Treatment – C.gigas Larvae RNA from yesterday

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.

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.

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.