Tag Archives: RNA

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.

Reverse Transcription – Herring RNA from 20091026

Performed an RT reaction on pooled herring gonad and liver mRNA from 20091026 for James Raymond at the UNLV. A single RT reaction was performed using 12.75uL (208ng) of the pooled gonad mRNA and 5uL (132.5ng) of the pooled liver RNA, according to our default MMLV (Promega) protocol. After reaction was completed, sample was stored @ -20C and then shipped to James Raymond on 20130214.

qPCR – DNased Manila Clam Larvae RNA (from August 2012 – Dave’s Notebook)

Performed qPCR on Dave’s manila clam larvae DNased RNA from August 2012 using EF1a primers (SR IDs: 1463, 1474).

Master mix calcs are here. https://docs.google.com/spreadsheet/ccc?key=0AmS_90rPaQMzdHc5amwzZzdDa1d0VXQzLVU0WkFTc0E

Plate layout, cycling params, etc can be found in the qPCR Report (see Results).

Positive control was pooled cDNA taken from Dave’s cDNA plate on 8/7/2012.

Results:

qPCR Data File(CFX96) http://eagle.fish.washington.edu/Arabidopsis/qPCR/CFX96/Roberts%20Lab_2012-10-26%2010-48-07_CC009827.pcrd

qPCR Report(PDF) http://eagle.fish.washington.edu/Arabidopsis/qPCR/CFX96/Roberts%20Lab_2012-10-26%2010-48-07_CC009827.pdf

Here’s a quick Google Spreadsheet summary highlighting samples that came up positive/negative.

https://docs.google.com/spreadsheet/ccc?key=0AmS_90rPaQMzdFFHb3YwWE01UG00TnY3OWo2cWx2UVE

Approximately half of the samples (~27) came up positive for still having gDNA in them.

There are three pCO2 treatments: 1000ppm, 750ppm, and 400ppm. There are six sampling dates: 7/29/2011, 8/2/2011, 8/9/2011, 8/12/2011. Currently, it is unknown when the Day 0 samples were collected. Have emailed Dave for deets.

There are only two dates (7/29/2011 and 8/5/2011) that have a full set of samples (i.e. 1000ppm, 750ppm and 400ppm) that exhibit DNA-free RNA. Will discuss with Steven on how to proceed.

UPDATE 20121031 – Dave emailed and indicated the experimented started on 7/27/2011. Additionally, the two sample sets that are complete are Day 2 and Day 7. Discussing with Steven, we have decided to run a few genes and see how the expression levels compare to the NGS data analysis for these samples. If the qPCR data supports the NGS data, then that information will be relayed to the BMC Genomics reviewers in response to their critiques. A copy of the manuscript is here(may not be publicly viewable). https://docs.google.com/document/d/1Ii1lODz2oThiyxZtHBblUEdzyhIVq92n8jkEjhkuuts/edit

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.

Reverse Transcription – DNased C.gigas Larval RNA from 20120427

Performed reverse transcription using random primers (Promega) diluted 1:100 (5ng/uL) with 175ng of DNased total RNA. Random primers were used because we will be targeting V.tubiashii RNA instead of eukaryotic RNA. Reverse transcription was performed with M-MLV Reverse Transcriptase (Promega) according to the manufacturer’s protocol. Calcs are here.

 

Created dilutions of all samples to 100ng/uL in a volume of 50uL in preparation for qPCR analysis. Calcs are here.

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 (#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.