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.

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.

qPCR – Tim’s adults gigas challenge DNased RNA (from 20091002)

Performed qPCR using q18s primers on DNased RNA (1:100 dilution to match final concentration of template after making cDNA). qPCR set up and plate layout are here.

gDNA dilutions were used as positive controls. gDNA = BB11 (0.49ug/uL) from 20090519. Used 5uL of 1:10, 1:100 and 1:000 dilutions.

Results: gDNA dilutions look good. However, some samples are definitely coming up before the 40 cycle mark. Will re-DNase treat these.

DNase Treatment – Tim’s adult gigas challenge RNA (from 20090930)

Used 5uL of RNA from each sample, brought samples up to 50uL with H2O and treated according to Ambion’s Turbo DNA-free kit. Transferred treated samples to a PCR plate to facilitate further manipulation of the samples. Will perform qPCR on these samples to make sure treatment worked.

qPCRs – Check gDNA contamination with EF1 & 18s primers in gigas gill RNA (from yesterday)

Ran qPCR on RNA to evaluate gDNA contamination in the samples. Dilutions of the RNA were made at 1:100, which would be the equivalent amount when making cDNA (1:25) and diluting the cDNA (1:4) prior to using in a qPCR rxn. qPCR set up is here with cycling params. Plate layout is here.

gDNA dilutions were used as positive controls. gDNA = BB11 (0.49ug/uL) from 20090519.

Results: Hello! I’m an idiot. Nothing amplified because the EF1 primers are designed to cross an intron/exon boundary, thus they can’t amplify gDNA. Need to use the 18s primers instead.

 

This is an exact duplicate of the earlier qPCR from today, but using the correct (18s) primers! Ran qPCR on RNA to evaluate gDNA contamination in the samples. Dilutions of the RNA were made at 1:100, which would be the equivalent amount when making cDNA (1:25) and diluting the cDNA (1:4) prior to using in a qPCR rxn. qPCR set up is here with cycling params except q18s primers are substituted instead of qEF1. Plate layout is here.

gDNA dilutions were used as positive controls. gDNA = BB11 (0.49ug/uL) from 20090519.

Results: All samples show gDNA contamination. Will DNase them

RNA Isolation – Tim’s adult gigas challenge samples

RNA was isolated using 500uL of TriReagent for all samples. Samples were resuspended in 100uL of 0.1%DEPC-H2O and spec’d. Samples stored in Tim’s “NAME OF BOX” box.

Results:

AC# = Air Control Sample

CC# = CO2 Control Sample

AV# = Air Vibrio Sample

CV# = CO2 Vibrio Sample

All the samples look really good, even those that exhibited dark coloration carried over from extraction. Will check for gDNA contamination.

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

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.

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.

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.