Tag Archives: mRNA

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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EtOH Precipitation – Rick’s trout Ribosomoal-depleted RNA for SOLiD WTK (from today)

The “control” and “poly I:C” samples prepared earlier today were EtOH precipitated in preparation for fragmentation.

Added the following to each sample:

  • 18uL 5M ammonium acetate
  • 1uL glycogen
  • 2 vols. of 100% EtOH (74uL)

Samples were incubated O/N @ -80C.

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Ribosomal-depleted RNA – Rick’s trout RBC samples for the SOLiD WTK

Prior to starting the procedure, 0.5uL of total RNA was removed from each sample (control, polyI:C), diluted to ~5ng/uL. 1.5uL of each of these was transferred to a 0.5mL snap cap tube for running on the PicoChip on the Bioanalyzer. These were stored @ -80C in the “Bioanalyzer Samples” box.

The remaining total RNA from Rick’s trout RBC (~15uL of the “control” and 20uL of the “polyI:C”) was treated with Invitrogen’s RiboMinus Kit, according to protocol. Samples were then processed following Invitrogen’s Modified RiboMinus Concentration Module, but samples were eluted with 20uL of H2O, instead of 30uL. Samples were spec’d.

Results:

The “poly I:C” sample looks good and gave a return of ~800ng, which is ~1% of the total starting RNA (20uL x 0.42ug/uL = 8.4ug). The “control” sample, however, is well short of the expected 1% yield. Recovery was ~220ng, which is only ~0.25% of the total starting RNA (15uL x 0.571ug/uL = 8.565ug). Will proceed to EtOH precipitate the samples in preparation for fragmentation.

Transferred 0.75uL of the “control” sample to a 0.5mL snap cap tube containing 0.75uL of H2O. Transferred 0.25uL of the “poly I:C” sample to a 0.5mL snap cap tube containing 1.25uL of H2O. Samples were stored @ -80C in the “Bioanalyzer Samples” box.

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

Was given 1ug of each of these two RNA samples and processed them with Promega’s PolyA Tract Kit according to protocol. After elution, the samples were EtOH precipitated @ -20C for 30mins, pelleted 30mins 16,000g for 30mins, 4C. Supe removed, RNA washed with 1mL 70% EtOH and spun 15mins 16,000g, 4C. Supe removed. Resusupended in 15uL of 0.1% DEPC-H2O and spec’d.

Results: No measurable amount of RNA in either sample. Samples were stored @ -80C.

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mRNA Isolation – hard clam gill #1 DNased RNA from today

DNased RNA from earlier today was split into four equal parts (175uL = 39.8ug). Three will be used for mRNA isolation and the fourth will remain as total RNA. Three of these were precipitated according to Ambion PolyAPurist Protocol: 1/10 volume 5M ammonium acetate, 1uL glycogen and 2.5 volumes of 100% EtOH. Incubated @ -80C for 30 mins. One sample was processed with the Promega PolyA Tract kit. The remaining two samples were processed according to PolyAPurist Protocol. Of those two, one of the samples was processed a second time to evaluate the effectiveness of running a sample through the PolyAPurist Protocol twice.

mRNA samples were precipitated O/N @ -20C according to the PolyAPurist Protocol.

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