Preprocessing and off scale data management

Here we discuss all kinds of problems related to primer-dimers and other off-scale measurements

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Preprocessing and off scale data management

Postby jdenk » Mon Nov 04, 2013 5:50 pm

Hey,

GenEx offers a lot of preprocessing steps in order to handle off scale data and to generate reliable CTs. In my experiment, i measured both -RT and NTC controls to asses gDNA and primer-dimers. I'm now thinking of how to put the preprocessing steps in order to obtain appropriate Ct values. The preprocessing steps i think to make sense are:

1. define overall Ct cut-off
2. replace missing data based on PCR replicates
3. define Ct standard deviation cut off to remove Cts with high variability
4. average PCR replicates
5. subtract gDNA by using -RT correcion of measured no enzyme controls
6. using the "negative control" option in the quality control section to set an individual cut-off (lets say 5 Cts) based on the negative control Ct values

Im using the high SD option to remove out of range data, since i am measuring only duplicates on the PCR levels - so a grubbs test cannot be applied!

Any comments are appreciated!

Cheers!
jdenk
 
Posts: 11
Joined: Fri Feb 22, 2013 11:27 am

Re: Preprocessing and off scale data management

Postby Mikael Kubista » Wed Nov 06, 2013 11:52 pm

Sounds good to me!
Mikael Kubista
 
Posts: 455
Joined: Tue Jul 01, 2008 12:28 pm


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