Tag Archives: Whole Transcriptome Analysis Kit

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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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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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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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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