DNase – Withering Syndrome RNA

Withering syndrome RNA, extracted from water filters, was confirmed via qPCR to contain detectable quantities of withering syndrome DNA. Before proceeding to make cDNA, residual gDNA carryover from the RNA extraction needs to be removed.

Performed DNase treatment with the Turbo DNA-free Kit (Ambion/Life Technologies), following the rigorous. Used 10μL of each of the following template RNA:

  • Day 0-1
  • Day 3-1
  • Day 7-1
  • Day 11-1

Reactions were performed in a volume of 50μL. Used 1μL of DNase for the first 30mins @ 37C and then added an additional 1μL of DNase for the final 30mins @ 37C. Samples were inactivated according to the manufacturer’s protocol and spec’d on the Roberts Lab NanoDrop1000.

Results:

Yields are consistent. The OD260/280 values are still poor (didn’t expect them to change, though). Will qPCR to verify removal of gDNA.

qPCR – Withering Syndrome RNA Check

Ran withering syndrome qPCR on the following RNA samples provided by Lisa to determine if it’s necessary to DNase treat them prior to performing reverse transcription:

I believe these were extracted from water filters collected from basement abalone tank water. The OD260/280 ratios are pretty bad, particularly for RNA, but will proceed anyway.

Master mix calcs are here: 20150318 – qPCR WSN RNA Test

Since I’m anticipating using 1000ng of RNA in the reverse transcription reactions (25μL reaction volume = 40ng/μL), I only used 1μL of RNA (~100ng), and positive control, template in each reaction to semi-accurately represent the quantity of RNA that would be loaded in a qPCR reaction with 2μL of cDNA.

All samples were run in duplicate. Positive control was the 3e6 p18RK7 standard curve sample from 20120731.

Results:

qPCR Report (PDF): Sam_2015-03-18 10-11-35_CC009827.pdf
qPCR Data File (CFX96): Sam_2015-03-18 10-11-35_CC009827.pcrd

All samples are positive for withering syndrome. Will proceed with DNase treatment.

qPCR – Evaluation of withering syndrome and phage presence in holding tanks

In anticipation of receiving a large quantity of abalone from Japan, Carolyn wants to assess  current infection status of our abalone to make decisions on how/where to house the incoming abalone.

Ran a qPCR to detect withering syndrome and the withering syndrome phage on the following DNA samples isolated today by Lisa:

  • RR1 (Haliotis discus discus) – Seawater DNA
  • RR2 (Haliotis diversicolor) – Seawater DNA
  • 14:5-1 – 4- Dg DNA

Positive control: pCR2.1/ORF25 (1:1000) from 20141008.

Primers used:

  • Withering Syndrome – WSN1F/R
  • Phage – 1_ORF25F_225_CSF, 1_ORF25R_399_CSF,

All samples were run in duplicate.

Master mix calcs are here: 20150316 – qPCR H.discus H2O and feces

Plate layout, cycling params, etc. can be viewed in the qPCR Report (see Results).

Results:
qPCR Report (PDF): Sam_2015-03-16 16-46-06_CC009827.pdf
qPCR Data File (CFX96): Sam_2015-03-16 16-46-06_CC009827.pcrd

Withering syndrome
– standard curve is perfect
– all samples, except seawater RR2, amplified

Phage
– no standard curve; this is not ready yet; as such, you can ignore the copy number (SQ) listed in the data file
– consistent amplification in both seawater samples (RR1, RR2)
– zero or inconsistent (i.e. one of two reps amplified) in remaining samples
– melt curves in the RR1 and RR2 samples exhibit multiple peaks, suggesting amplification of multiple targets (i.e. lack specificity)
– melt curves in the remaining samples only exhibit single peaks
– RR2 melt curves are shifted 2C (82C), compared to all other samples (80C)

qPCR – 2011 Ab Endo Water Filters

Master mix calcs are here: 20141104 – qPCR 2011 Ab Endo Water Filters

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

Standard curve was p18RK7 from 20120731.

Baseline threshold set to 580, based on calculations by Lisa.

Results:

qPCR Report (PDF): Sam_2014-11-04 14-25-07_CC009827.pdf
qPCR Data File (CFX96): Sam_2014-11-04 14-25-07_CC009827.pcrd

All data was entered in to the Ab Endo Samples spreadsheet. Samples that require repeating were highlighted in orange.

qPCR – 2011 Ab Endo Water Filters

Master mix calcs are here: 20141021 – qPCR 2011 Ab Endo Water Filters

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

Baseline threshold set to 580, based on calculations by Lisa.

Results:

qPCR Report (PDF): Sam_2014-10-21 10-42-19_CC009827.pdf

qPCR Data File (CFX96): Sam_2014-10-21 10-42-19_CC009827.pcrd

All data was entered in to the Ab Endo Samples spreadsheet. Samples that require repeating were highlighted in orange.

qPCR – 2011 Ab Endo Water Filters

Master mix calcs are here: 20141016 – qPCR 2011 Ab Endo Water Filters

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

Baseline threshold set to 580, based on calculations by Lisa.

Results:

qPCR Report (PDF): Sam_2014-10-20 11-08-57_CC009827.pdf

qPCR Data File (CFX96): Sam_2014-10-20 11-08-57_CC009827.pcrd

All data was entered in to the Ab Endo Samples spreadsheet. Samples that require repeating were highlighted in orange.

qPCR – 2011 Ab Endo Water Filters

These are repeats of samples that did not have amplification in both replicates when previously run.

Master mix calcs are here: 20141016 – qPCR 2011 Ab Endo Water Filters

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

Baseline threshold set to 580, based on calculations by Lisa.

Results:

qPCR Report (PDF): Sam_2014-10-16 09-15-49_CC009827.pdf

qPCR Data File (CFX96): Sam_2014-10-16 09-15-49_CC009827.pcrd

All data was entered in to the Ab Endo Samples spreadsheet. Samples that require repeating were highlighted in orange.

qPCR – 2011 Ab Endo Water Filters

Master mix calcs are here: 20140925 – qPCR 2011 Ab Endo Water Filters_02

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

Baseline threshold set to 580, based on calculations by Lisa.

Results:

qPCR Report (PDF): Sam_2014-09-25 09-46-55_CC009827.pdf

qPCR Data File (CFX96): Sam_2014-09-25 09-46-55_CC009827.pcrd

All data was entered in to the Ab Endo Samples spreadsheet. Samples that require repeating were highlighted in orange.

qPCR – Ab Endo 2011 Water Filters Withering Sydnrome

Ran qPCR on subset of Ab Endo 2011 water filters extracted on 20140822 using the withering syndrome qPCR assay. See Ab Endo Samples spreadsheet and/or the qPCR Report (see Results below) for which samples were qPCR’d.

Master mix calcs are here: 20140826 – qPCR 2011 Ab Endo Water Filters_01

All samples were run in duplicate.

Standard curve was p18RK7 from 20120731.

Used ABI probe.

Plate layout, cycling params, etc can be seen in the qPCR Report (see Results below).

Results:

qPCR Report (PDF): Sam_2014-08-26 15-01-50_CC009827.pdf

qPCR Data File (CFX96): Sam_2014-08-26 15-01-50_CC009827.pcrd

Threshold was set at 580 RFUs, based on an average of runs using Promega 2x Probe Master Mix by Lisa.

qPCR – Ab Endo 2011 Water Filters Withering Syndrome

Ran qPCR on subset of Ab Endo 2011 water filters extracted on 20140822 using the withering syndrome qPCR assay. See Ab Endo Samples spreadsheet and/or the qPCR Report (see Results below) for which samples were qPCR’d.

Master mix calcs are here: 20140826 – qPCR 2011 Ab Endo Water Filters_01

All samples were run in duplicate.

Standard curve was p18RK7 from 20120731.

Used IDT probe.

Plate layout, cycling params, etc can be seen in the qPCR Report (see Results below).

Results:

qPCR Report (PDF): Sam_2014-08-26 16-31-58_CC009827.pdf

qPCR Data File (CFX96): Sam_2014-08-26 16-31-58_CC009827.pcrd

Threshold was set at 580 RFUs, based on an average of runs using Promega 2x Probe Master Mix by Lisa.

Well, this is not good. Curve looks bad. Only difference is using the IDT probe. But, there’s not sufficient ABI probe to handle the remainder of the samples. Will have to test out some things with the IDT probe…