Tag Archives: trout

Reverse Transcription/cDNA purification/Emulsion PCR – Ligation rxns of trout fragmented RNA for SOLiD WTK (from yesterday)

The four samples from yesterday were prepared according to the Agilent SOLiD WTK protocol. Briefly:

 

 

Results: All four samples appear to have cDNA. Interestingly, the “Amped cDNA trout RBC control ribo(-)” sample was the sample that had no detectable RNA after fragmentation, BUT this sample produced the highest yield of cDNA… See below.

1.5uL of each sample was transferred to a 0.5mL snap cap tube and stored @ -80C in the “Samples for Bioanalyzer” box for submission on the DNA 1000 Chip.

The Yellow/Brown plot above is the “Amped cDNA trout RBC poly I:C ribo(-) & polyA” sample and exhibits a strange profile at the 220-230nm range that differs than the three other samples.

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Adapter Ligation – Rick’s trout fragmented control/poly I:C samples for SOLiD WTK

See the Next Gen Seq Library Database for more info. Processed the 4 samples (one set Ribominus only, one set Ribominus + PolyA enriched) according to the Agilent WTK. Briefly:

  • Speedvac’d samples to dryness
  • Resuspended RNA in 3uL H2O
  • Adapter rxn. Used all 3uL of RNA (used only 1uL of RBC Ribo only sample due to high concentration)
  • Ligation rxn

Incubated 16C for 16hrs.

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Bioanalyzer Submission – Rick’s trout RBC samples (various dates)

Submitted Rick’s trout RBC samples to FHRC for bioanalysis using the PicoChip for use with the SOLiD WTK. Submission sheet is here.

Results: Received 20091001.

Lanes 1 & 2 = ribo-depleted AND polyA enriched

Lanes 3 & 4 = ribo-depleted only

Lanes 5 & 6 = total RNA

Lanes 7 & 8 = ribo-depleted only

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RNA Fragmentation – Rick’s trout RBC samples prepped earlier today

EtOH Precipitaiton – Rick’s trout Ribosomoal-depleted RNA for SOLiD WTK (continued from yesterday)

Continued precipitation. Spun samples 30 mins, 16,000g, 4C. Removed supe. Added 1mL 70% EtOH. Spun samples 15mins, 16,000g, 4C. Removed supe. Resuspended in 8uL H2O. Proceeded with SOLiD WTK fragmentation.

 

RNA Fragmentation

Samples were fragmented according to the Whole Transcriptome Kit protocol. Samples were then cleaned up using Invitrogen’s RiboMinus Concentration Module, according to SOLiD WTK protocol. Briefly:

  • Added 1X volume of binding buffer (100uL)
  • Added 100% EtOH (250uL)
  • Eluted with 20uL of H2O.

Samples were spec’d.

Results:

Control Sample – Virtually nothing there. Hopefully it’s just too dilute for the NanoDrop, however I have a feeling this sample is bad (degraded?) 1.5uL of the sample has been transferred to a 0.5mL snap cap tube to send off for the Bioanalyzer.

Poly I:C Sample – Looks great, excellent recovery. 0.25uL of this sample was transferred to a 0.5mL snap cap tube containing 1.25uL of H2O to send off for the Bioanalyzer.

Samples were stored @ 80C until resutls from the Bioanalyzer are received.

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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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RNA Fragmentation – Rick’s trout RBC samples prepped earlier today (see below)

Samples were fragmented according to the Whole Transcriptome Kit protocol. Samples were then cleaned up using Invitrogen’s Modified RiboMinus Concentration Module, according to protocol. Briefly:

  • Added 1X volume of binding buffer (100uL)
  • Added equal volume of 100% EtOH so that [EtOH] = 50% (200uL)

Followed remainder of protocol and eluted with 20uL of H2O. Samples were spec’d.

Results:

Assuming the NanoDrop readings are accurate (according to the Whole Transcriptome Kit, these may NOT be accurate), got yields of ~93ng for the RBC Control sample and ~132ng for the RBC poly1:C sample. Transferred 1.5uL of each sample to separate 0.5mL tubes for submission to the Bioanalyzer. These were stored @ -80C in the “Bioanalyzer Samples” box. The remainder of the samples were stored @ -80C in the “Samples from Other Researchers” 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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