Data Analysis – RLOv DNA Helicase qPCR Limit of Detection Calcs

I previously ran three qPCR plates (20160121, 20160122, & 20160125) with 20 reps of each of the following copy numbers to determine the limit of detection (or, analytical sensitivity) of this qPCR assay: 30, 10, 3, 1

Additionally, I determined the average baseline threshold to set (580.5), based on eight qPCRs (see 20160128).

Performed calculations and determined limit of detection (defined as >95% of samples produced amplification) for this assay is three copies. The single copy sample amplified 88% of the time. This also means that our cycle threshold cut-off for this qPCR assay should be 37.9, as the mean Cq for three copies was 37.33, with a standard deviation of 0.53.

The data and calculations can be seen below (Google Sheet – scroll to the right to see calcs):

Google Sheet: RLOv_DNA_helicase_LoD_calcs

 

 

Data Analysis – qPCR Baseline Threshold Determination for RLOv DNA Helicase qPCR Assay

Idetermined a qPCR baseline threshold to use for the withering syndrome RLOv DNA helicase qPCR assay in the following fashion.

I adjusted baseline threshold values of the following eight qPCR runs so that the mean Cq  values for each component of the standard curves (per plate) across plates were within 0.5 Cqs and averaged the baseline thresholds.

The average baseline threshold was 580.5.

This value will be manually applied to all past and future RLOv DNA helicase qPCR runs that were conducted on the Friedman Lab CFX96.

FILE ADJUSTED THRESHOLD
Sam_2016-01-25 10-48-06_CC009827.pcrd 609
Sam_2016-01-22 10-15-55_CC009827.pcrd 569
Sam_2016-01-21 15-12-31_CC009827.pcrd 576
Sam_2016-01-06 16-01-36_CC009827.pcrd 550
Sam_2015-12-28 15-44-35_CC009827.pcrd 613
Sam_2015-12-28 11-20-16_CC009827.pcrd 614
Sam_2015-12-24 11-02-59_CC009827.pcrd 531
Sam_2015-12-23 13-57-01_CC009827.pcrd 582

 

qPCR – Promega 2x GoTaq Probe Master Mix Withering Syndrome Assay Limit of Detection

Ran limit of detection qPCR for the Promega 2x GoTaq Probe Master Mix using p18RK7 (from 20120730) primary curve and p18RK7 low curve (from 20140507).

Master mix calcs are here: 20140506 – qPCR WSN1 Promega LoD-1

Cycling params, plate layout, etc. can be found in the qPCR Report in the Results below.

Results:

qPCR Report (PDF): Sam_2014-05-21 14-04-07_CC009827.pdf
qPCR Data File (CFX96): Sam_2014-05-21 14-04-07_CC009827.pcrd

Data can be found here: Promega Master Mix Withering Syndrome Limit of Detection

qPCR – Promega 2x GoTaq Probe Master Mix Withering Syndrome Assay Limit of Detection

Ran limit of detection qPCR for the Promega 2x GoTaq Probe Master Mix using p18RK7 (from 20120731) primary curve and p18RK7 low curve (from 20140507).

Master mix calcs are here: 20140506 – qPCR WSN1 Promega LoD-1

Cycling params, plate layout, etc. can be found in the qPCR Report in the Results below.

Results:

qPCR Report (PDF): Sam_2014-05-21 10-07-34_CC009827.pdf
qPCR Data File (CFX96): Sam_2014-05-21 10-07-34_CC009827.pcrd

Data can be found here: Promega Master Mix Withering Syndrome Limit of Detection

NOTE: Well B6 is an outlier. Without that sample, the R^2 changes from 0.977 to 0.990. The Cq value for this sample has been highlighted in its entry on the Promega Master Mix Withering Syndrome Limit of Detection spreadsheet.

qPCR – Promega 2x GoTaq Probe Master Mix Withering Syndrome Assay Limit of Detection

Ran limit of detection qPCR for the Promega 2x GoTaq Probe Master Mix using p18RK7 (from 20120731) primary curve and p18RK7 low curve (from 20140507).

Master mix calcs are here: 20140506 – qPCR WSN1 Promega LoD-1

Cycling params, plate layout, etc. can be found in the qPCR Report in the Results below.

Results:

qPCR Report (PDF): Sam_2014-05-20 13-17-13_CC009827.pdf
qPCR Data File (CFX96): Sam_2014-05-20 13-17-13_CC009827.pcrd

Data can be found here: Promega Master Mix Withering Syndrome Limit of Detection

qPCR – p18RK7 Low Curve Check

I needed additional dilutions for a low curve to finish validating the Promega 2x GoTaq Probe Master Mix limit of detection. Will test the low curve before continuing the full limit of detection process.

Master mix calcs are here: 20140507 – qPCR p18RK7 low curve check

Results:

qPCR Report (PDF): Sam_2014-05-07 14-47-55_CC009827.pdf
qPCR Data File (CFX96): Sam_2014-05-07 14-47-55_CC009827.pcrd

New low curve looks good. Will use this for subsequent limit of detection tests.

qPCR – Promega 2x GoTaq Probe Master Mix Withering Syndrome Assay Limit of Detection

Now that we’ve decided to go with the Promega master mix, we need to re-do the limit of detection to validate the assay, as far as sensitivity goes.

Master mix calcs are here: 20140506 – qPCR WSN1 Promega LoD-1

Plate layout, cycling params, etc can be found in the qPCR Report in the Results below.

Results:

qPCR Report (PDF): Sam_2014-05-06 14-38-39_CC009827.pdf
qPCR Data File (CFX96): Sam_2014-05-06 14-38-39_CC009827.pcrd

Data can be found here: Promega Master Mix Withering Syndrome Limit of Detection

qPCR – Withering Syndrome qPCR Assay Validation: Reproducibility (CFX96)

Ran additional reps of the Low and Medium Tissue samples, due to poor replication in an earlier run on these samples.

Tissue

  • Low: 09:16-18
  • Med: 09:16-22

Master mix calcs are here.

Baseline threshold was set to 400 and cycles to analyze was set to 41.

Results:

qPCR Data File (CFX96)

qPCR Report (PDF)

qPCR – Withering Syndrome qPCR Assay Validation: Reproducibility (Opticon 2)

Run on the Roberts’ Lab Opticon 2 as part of the reproducibility aspect of assay validation. Master mix calcs are here.

Standard curve was the p16RK7 NcoI-linearized curve made on 20120730.

Baseline threshold was set to 400 and cycles to analyze was set to 41.

Results:

qPCR Data File (Opticon 2)

qPCR Report (Google Spreadsheet)

Things looked good. Will have Lisa proceed with her part of the reproducibility assay validation.