qPCR – Withering Syndrome Phage

Ran qPCR using the following primer sets designed off of the potential WS phage sequence that Stan Langevin has sequenced:

  • 1_ORF25F_225_CSF, 1_ORF25R_399_CSF
  • 2_ORF25_121_CAB, 2_ORF25R_320_CAB
  • 3_ORF20F_121_CSF, 3_ORF20R_326_CSF

Templates tested were abalone digestive gland gDNA:

  • 06:6-41 (4/7/2008) – Positive for withering syndrome and phage; used in the MiSeq run by Stan Langevin.
  • 06:6-53 (4/9/2008) – Positive for withering syndrome and phage; used in the MiSeq run by Stan Langevin.
  • 08:4-1 – Positive for withering syndrome only – no phage.
  • UW08:22-11A – Pinto abalone naive for both withering syndrome and phage.

Master mix calcs are here: 201400820 – qPCR WS phage

All samples were run in duplicate. See qPCR Report (see Results) for plate layout, cycling params etc.

Results:
qPCR Report (PDF): Sam_2014-08-20 14-29-56_CC009827.pdf
qPCR Data File (CFX96): Sam_2014-08-20 14-29-56_CC009827.pcrd

Great news! No amplification in the 08:4-1 (positive for WS, but naive for phage)!! These results strongly suggest that these primers are specific for the WS bacteriophage! This is really cool and exciting. Next steps will be to confirm via in-situ hybridization (ISH).

Additional summary of the results:

Primer set ORF25_CSF shows the highest sensitivity.

Primer set ORF20_CSF fails to amplify anything in 06:6-53

DNA Quanting – Black Abalone Digestive Gland DNA

Quantified gDNA from the following samples in preparation for high-throughput sequencing by Stan Langevin’s group. Quantification was done using Pico Green, Tecan plate reader and Magellan 6 software. The r^2 value of the standard curve was 0.9986 and replicates showed little variation.

Concentrations below are the mean of three replicates and are in ng/uL.

  • 06:5-28 – 42.4
  • 06:6-41 – 60.3
  • 06:6-44 – 57.1
  • 06:6-53 – 72.1
  • 06:6-54 – 40.1
  • 06:6-55 – 40.5
  • 06:6-66 – 40.6

Stan Langevin has requested at least 50ng of DNA from each sample. I will aliquot ~100ng of each sample into individual tubes. He will be picking up aliquots tomorrow morning for sequencing on the MiSeq (Illumina).

RNA Isolation – Black Abalone DG Withering Syndrome Only (WSO)

Isolated RNA from 3 digestive gland (DG) samples: 08:13-7, 08:13-9, and 08:13-16 using the RNA Power Soil Kit (MoBio) according to the manufacturer’s protocol and spec’d on the Roberts Lab NanoDrop1000.

Results:

RNA is very high quality. Will prepare for submission for Illumina HiSeq sequencing. Sample pooling info is here.

RNA Isolation & DNase – Abalone Dg

Isolated RNA from Red Abalone Dg (10:25-24, 25, 26) using TriReagent, according to protocol. These samples are expected to have high levels of the Rickettsia phage that I’ve been attempting to investigate. The tissue samples were taken on 20110224 by Lisa and stored in RNA Later @ 4C. Remaining tissue will be returned to existing tubes and stored @ -80C. Previously, RNA from Ab Dg had been isolated using the MoBio RNA PowerSoil Kit due to its ability to produce extremely clean RNA from Dg (a nasty tissue). However, we currently do not have that kit. RNA was resuspended in 100uL of 0.1% DEPC-H2O and spec’d on the Roberts Lab ND1000. RNA will be stored in “Sam’s -80C Box #1″ in the Roberts’ Lab -80C freezer.

Results:

As expected, the RNA quality is low (based on OD260/280), as the TriReagent method of isolation does not clean up the RNA as extensively as the MoBio RNA PowerSoil Kit which has been previously used. Additionally, the RNA solution is brown colored. Will DNase RNA.

 

DNase – Abalone Dg RNA (from earlier today)

DNased Abalone Dg RNA that was isolated earlier today using Ambion’s Turbo DNA-free Kit following the “rigorous” protocol. Briefly, 5ug of RNA was brought up to 45uL with nuclease-free H2O. Reaction was brought to 50uL with 10x DNase Buffer and 1uL of DNase. Sample was incubated @ 37C for 30mins. An additional 1uL of DNase was added, mixed and incubated for 30mins. DNase was inactivated, supe transferred to clean tube and spec’d on Roberts’ Lab ND1000. DNased RNA will be stored in “Sam’s RNA Box #1″ in the Roberts’ Lab -80C freezer.

Results:

OD260/280 values still look poor (which was expected based on initial isolation from earlier today). Will check DNased RNA for residual gDNA carryover.

Reverse Transcription – Abalone Dg for Virus Primer Optimization

Performed reverse transcription of 5 black abalone Dg DNased RNA (from 20090421) according to Promega’s recommendations using M-MLV reverse transcriptase. Calculations are here.

DNased RNA Samples Used

Sample Name New Score
07:12-17 3
08:3-6 2.5
08:3-18 1
08:4-1 0
08:4-2 0

In order to have sufficient cDNA to use for primer optimization, the 07:12-17 and the 08:3-6 cDNA samples were pooled.

Samples were stored @ -20C in Sam’s cDNA Box #2 (Roberts Lab).