[BioC] HTqPCR question

[Simon Melov]

I'm trying to use the package developed by Heidi Dvinge (HTqPCR), and I've a couple of basic questions. The package seems very nice, however, I'm using a high throughput real-time PCR system, the Biomark from Fluidigm, which can carry out upto 40,000 PCR's in a single run (approximately). Most of my runs use about 10,000 PCRs, with multiple technical replicates, and occasionally I use several plates if I'm assying many samples, and dozens of genes.

Typically, I've been using the geNorm approach for identifying potential normalizers in a suite of genes in any given biological paradigm, as a priori its always best to identify a gene which doesnt change in the question of interest (as opposed to using something like actin, and assuming it doesnt change). Is there a way of identifying the "best" normalizer in HTqPCR? Also, I'm having trouble specifying flags. I understand how to designate the appropriate column in the file as the flag column, however, I dont understand how to specify the pass/fail criteria. In the Biomarks output, each PCR reaction is specified as "Pass" or "Fail". I've been over the vignette for HTqPCR, and cant see how to define the "Faliure" criteria.

In addition, I'm a bit fuzzy on how HtQPCR handles technical reps as opposed to biological reps. There is some discussion of this in the vignette, but I'm having trouble following it.

Here are a few lines of our typical output from our Biomark machine in case this helps.


Chip Run Filename

X:\Real-time studies\Biomark\HK_Mar2010\ChipRun.bml

















Application Version

2.0.2

















Application Build

20080502.1530

















Export Type

Table Results

















Quality Threshold

0.65

















Baseline Correction Method

Linear

















Ct Threshold Method

Auto (Detectors)

























































Experiment Information

Experiment Information

Experiment Information

Experiment Information

Experiment Information

Experiment Information

FAM-MGB

FAM-MGB

FAM-MGB

FAM-MGB

Chamber

Sample

Sample

Sample

FAM-MGB

FAM-MGB

Ct

Ct

Ct

Ct

ID

Name

Type

rConc

Name

Type

Value

Quality

Call

Threshold

S48-A01

T4_12D

Unknown

1

T09F3_3_HK

Test

19.0089363455255

0.97

Pass

0.030362744680761

S48-A02

T4_12D

Unknown

1

T09F3_3_HK

Test

16.9882713869506

0.98

Pass

0.030362744680761

S48-A03

T4_12D

Unknown

1

F33H1_2_HK

Test

18.6642291753142

0.92

Pass

0.0291832001753495

S48-A04

T4_12D

Unknown

1

F33H1_2_HK

Test

18.8928045555981

0.91

Pass

0.0291832001753495

S48-A05

T4_12D

Unknown

1

K10B3_8_HK

Test

20.498770043292

0.98

Pass

0.0433122130336323

S48-A06

T4_12D

Unknown

1

K10B3_8_HK

Test

20.2049170952598

0.99

Pass

0.0433122130336323





Any help would be greatly appreciated!

thanks

Simon.