Tag Archives: WB

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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