Tag Archives: mRNA enrichment

mRNA Isolation – Pooled Black Abalone Dg RNA (from Abalone Dg Exp 1)

mRNA was isolated for SOLiD sequencing by HTGU. Made two pools of San Nick RNA (Control and Exposed) using equal amounts (5ug) of each individual sample. Individual samples used can be found here. mRNA was isolated using Ambion’s Micro PolyAPurist Kit according to protocol. Procedure was performed two times on each pool and then EtOH precipitated. Samples were resuspended in 10uL of The RNA Storage Solution provided in the kit and spec’d on the Roberts Lab ND1000. Samples were stored @ -80C in the “Next Gen Sequencing Libraries” box.

Results:

Yields are pretty good from both samples (~500ng). However, the OD260/280 values are rather poor.

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RNA Precipitation & mRNA Isolation for SOLiD Libraries – Pooled abalone total RNA: Carmel control, Carmel exposed

RNA of 8 samples from each group was pooled equally from each individual. RNA was precipitated according to Ambion’s MicroPolyA Purist Kit. Used 0.1 volumes of 3M NaAOc, pH=5.2, 2.5vols of 100% EtOH and incubated 30min @ -80C. Pelleted RNA 16,000g, 30mins. Washed pellet w/70% EtOH and pelleted RNA 16,000g, 15mins. Pellets were resuspended in 50uL nuclease-free H2O and spec’d:

Total RNA pools look really nice. ~45ug of total RNA in each sample.

Isolated mRNA from each pool using Ambion’s MicroPolyA Purist Kit according to protocol. Samples were processed 2x as recommended by Ambion’s SOLiD Whole Transcriptome Analysis Kit. Final elution was 200uL of The RNA Storage Solution. Samples were spec’d:

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mRNA Precipitation for SOLiD – Perch, Lake Trout, & Herring mRNA (CONTINUED from yesterday)

mRNA was pelleted and washed according to Ambion’s MicroPolyA Purist Kit. Pellets were resuspended in 8uL nuclease-free H2O and spec’d. 0.5uL was taken from each sample, transferred to a fresh tube, diluted to ~5ng/uL and stored @ -80C for eventual Bioanalyzer analysis. mRNA samples were stored @ -80C until we receive the Ribominus Concentration Module Kit from Invitrogen (turns out we didn’t have any!) for cleaning up the RNA after fragmentation.

Results:

Overall, this mRNA doesn’t look that great. However, I did notice that all samples had (to varying degrees) particulate matter that wouldn’t dissolve. Prior to spec’ing, the particulate matter was pelleted so as to not interfere. All samples will continue to be prepped for SOLiD analysis despite poor 260/280 ratios and low yields.

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mRNA Isolation for SOLiD – Perch, Lake Trout, and Herring total RNA

Received pooled lean and siscowet RNA from Rick. Samples will be processed immediately for SOLiD fragment libraries. Two 1.5mL snap cap tubes labelled:

L.T. 2ug muscle sisco pool

L.T. 2ug muscle lean pool

RNA was first precipitated according to the Ambion MicroPolyA Purist Kit protocol (0.1 vol 5M ammonium acetate, 1uL glycogen, 2.5 vols 100% EtOH). Samples were incubated @ -80C for 30mins. Samples were resusupended in 250uL nuclease-free H2O and spec’d.

 

Starting quantities PRIOR to total RNA precipitation:

Perch Samples:

WB tRNA (WB tRNA ~12ug 24.5uL)

PQ tRNA (PW tRNA ~12ug 21.88uL)

CT tRNA (CT tRNA ~12ug 25uL)

Herring:

1 G/O HPWS09 (20ug)

Lake Trout:

L.T. 20ug muscle sisco pool

L.T. 20ug muscle lean pool

Removed 1uL of each sample, diluted to ~5ng/uL and stored @ -80C to run on the Bioanalyzer.

Used Ambion MicroPolyA Purist Kit according to protocol. Samples were treated twice to ensure elimination of rRNA from the samples. After second run through MicroPolyA Purist, samples were EtOH precipitated O/N @ -20C according to Ambion’s MicroPolyA Purist protocol.

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RNA Fragmentation – Herring Liver mRNA for SOLiD Libraries

Samples from 20091203. 0.5uL was removed from each and transferred to separate tubes and diluted to < 5ng/uL for subsequent Bioanalyzer analysis using the Pico chip. Samples were fragmented using RNase III according to the Ambion WTK protocol and then cleaned up/concentrated using the Invitrogen RiboMinus Concentration Module according to the Ambion WTK protocol.

Samples were spec’d prior to running on the Bioanalyzer:

Concentrations/absorbance values are not accurate when using the NanaDrop after using the RiboMinus Concentration module, according to the Ambion WTK protocol. However, yields seem pretty good…

Total, mRNA and fragmented mRNA from each of the four samples was run on the Pico chip with the Eukaryote Total RNA Bioanalyzer protocol.

Results:

The 2L tot (total RNA) and 3L tot (total RNA) samples are clearly very good quality. 2L tot does exhibit some very slight degradation, though. 4L tot (total RNA) and 6L tot (total RNA) show a much greater degree of degradation. All mRNA samples show complete removal of any trace, contaminating rRNA. The fragmented samples (the last four samples on the gel image above) all appear to be perfect. The 4L frag sample simply has less RNA loaded and that is why it is not as dark as the other three fragmented samples. Despite the degradation in the 4L tot and 6L tot samples, the fragmentation profile looks good and we will proceed with making the cDNA libraries for those samples.

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RNA Precipitation – Herring Liver RNA for SOLiD Libraries (continued from yesterday)

RNA Precipitation

Samples were pelleted for 30mins, 16,000g @ 4C. Supe was discarded, samples quick spun, residual supe removed and then washed with 1mL 70% EtOH. Tubes were vortexed until pellet came off of bottom of tube and then spun 15mins, 16,000g @ 4C. Supe was discarded, samples quick spun, residual supe removed and then resuspended in 250uL of 0.1% DEPC-H2O in preparation for mRNA isolation using the Ambion Micro PolyA Purist Kit.

 

mRNA Isolation

Clean RNA from earlier today was processed according to the Ambion Micro PolyA Purist Kit to isolate mRNA. This procedure was done two times to ensure full mRNA enrichment of the samples. Samples were then spec’d.

Results:

Yields:

3L – 10.66 ng/uL x 200uL = 2.132ug

6L – 6.97 ng/uL x 200uL = 1.394ug

2L – 10.89 ng/uL x 200uL = 2.178ug

4L – 6.43 ng/uL x 200uL = 1.286ug

 

mRNA Precipitation

Precipitation of mRNA from earlier today in preparation for fragmentation. Fragmentation requires mRNA volumes of <8uL, so after precipitation I will resuspend pellets in 8uL of 0.1% DEPC-H2O. Will use 1ug (this means 1/2 of 3L and 2L & all of 6L and 4L) of mRNA from each sample for precipitation. Remainder of 3L and 2L samples were stored @ -80C in “Herring RNA Box #1.”Samples were precipitated by adding 0.1 volumes 5M ammonium acetate, 1uL glycogen and 2 volumes of 100% EtOH. Samples were incubated O/N @ -20C.

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mRNA Isolation – Herring gonad/ovary RNA (from 20091023)

RNA Precipitation

Sample was spun 16,000g, 30mins, 4C. Supe removed. Pellet washed with 1mL 70% EtOH. Spun 16,000g, 10mins, 4C. Supe removed. Pellet resuspended in 250uL of nuclease free H2O. Will proceed with mRNA isolation.

 

mRNA Isolation

Isolated mRNA using Ambion’s MicroPolyA Purist Kit according to protocol. Performed two rounds of isolation to decrease residual rRNA carryover that we frequently see after a single round.

Results:

Started with ~90ug of total RNA. Yield of mRNA = 3.26ug. That is a ~3.6% recovery of mRNA.

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mRNA Isolation – Herring Liver RNA (from 20091021)

Isolated mRNA using Ambion’s MicroPolyA Purist Kit according to protocol. Performed two rounds of isolation to decrease residual rRNA carryover that we frequently see after a single round.

Results:

Started with ~500ug. Total yield = 5.3ug. That is a 1.06% recovery of mRNA.

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mRNA Isolation – Rick’s trout RBC samples previously treated with Ribominus Kit (by Mac)

Was given ~0.5ug of each of these two RNA samples and processed them with Ambion’s microPolyA Purist Kit according to protocol. After elution, the samples were EtOH precipitated @ -80C for 30mins, pelleted 30mins 16,000g for 30mins, 4C. Supe removed, RNA washed with 1mL 70% EtOH and spun 10mins 16,000g, 4C. Supe removed. Resusupended in 8uL of The RNA Storage Solution and spec’d.

Results:

Yield of ~320ng for RBC Control sample and ~360ng for RBC poly1:C sample. Will proceed to Whole Transctiptome Kit fragmentation step.

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mRNA Isolation – Gigas BB and DH samples previously treated with Ribominus Kit (by Mac)

Was given ~0.5ug of each of these two RNA samples and processed them with Ambion’s microPolyA Purist Kit according to protocol. After elution, the samples were EtOH precipitated @ -80C for 30mins, pelleted 30mins 16,000g for 30mins, 4C. Supe removed, RNA washed with 1mL 70% EtOH and spun 10mins 16,000g, 4C. Supe removed. Resusupended in 10uL of The RNA Storage Solution and gave back to Mac.

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