qPCR of environmental data

Here we discuss the use and processing of inter-plate calibrators

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qPCR of environmental data

Postby esauritorix » Tue Jul 22, 2008 11:22 am

I have about 12 plates with approximately 390 environmental DNAs, each plate has 1-7 dilutions of a standard DNA (pure DNA from a culture collection). I would like to normalize the standards for an inter-plate calibration that I would use to quantify more precisely all the environmental data. The problem is that I do not know what to do first with genex, shall I put all my data (1 column with well name and 1 column with Ct values ... any other value?) in an excel file and then import it in GenEx? I have treated individually all my values and no other corrections on the MXpro software have been selected. Once I have all the standards normalized I presume I'll have a normalized efficiency for the quantification of all the samples, won't I? How can I then tell Genex to use it to quantify the unknown samples in all different plates?
In addition I would like to ask you if I can analyze all my data from a catchment using 3 different methodologies (qPCR in SYBR green, qPCR in Taqman using 2 distinct marker genes) and comapare them. I have used a different dilution set for the SYBR green so I do not know if I have to repeat all the experiments with the same standard, the DNA is the same but I only have 3 dilutions in some experiments and from a different batch.

thanks,
Marco
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Re: qPCR of environmental data

Postby Mikael Kubista » Tue Jul 22, 2008 4:59 pm

Hello Marko,
I assume that every plate contains standard samples, which are used to construct a standard curve, and some test samples of unknown concentrations. The concentrations of these unknowns are readily estimated with GenEx using reverse calibration (which is the proper name for Absolute calibration). The approach is presented in the tutorial “Absolute quantification” available on: http://www.multid.se/demonstration.html.

Some comments.

Your data are measured in duplicates. Are these duplicates independent samples (sampling/biological replicates) or are they qPCR/RT replicates (technical replicates)? If they are technical replicates they shall be averaged during pre-processing and averaged values shall be used to construct the standard curve and to estimate unknowns. If they are sampling/biological replicates they shall be treated as independent samples.

Don’t forget to change the index of the unknown samples to “test” using GenEx Data Manager!

Since your concentrations are in copy numbers you must tick the box “Apply log on [x] in the “Reverse Calibration” control window in GenEx.

If the replicates were different samplings you should tick the box “Repeats exist” in the “Reverse calibration” control window and select the classification column that indexes the replicates (#Replicate).
Finally, if all plates are measurements of the SAME pathogen and you want to normalize them to perform a single large analysis, best way to do that is to normalize with the INTERCEPT that GenEx calculates for the independent standard curves. Only the Intercept – not the slope – is affected by the instrument setting. You can create an artificial sample where you enter the intercepts as CT values and use this for interplate calibration.

If the plates were analyzing different pathogens you cannot perform a single large analysis based on a common standard curve. But you can still merge the data into a single mdf file. When analyzing the data you just have to select the appropriate pathogen, and the corresponding classification columns with concentrations and repeat indexes. But since you are not gaining anything by having all the data in one file, you may as well keep them in separate files.

Good luck!
Mikael Kubista
 
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Re: qPCR of environmental data

Postby Mikael Kubista » Tue Jul 22, 2008 5:03 pm

Well, in essence, if the standards and targets are matched you shall be able to compare the results; in fact, the three approaches should give the same result if I am not misunderstanding you. I also assume that when you say “marker genes” you are referring to genomic PCR not RT.

Good luck!
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Re: qPCR of environmental data

Postby Branzila » Tue Feb 03, 2015 2:36 pm

I am screening 8 samples with many different genes on multiple plates. On each plate I ran two duplicate reactions with one control sample on one control gene as my interplate calibrator (two wells on each plate). How do I enter the calibrator CTs into the data matrix so that I can run interplate calibrator normalization using the data editor tool on Genex?
FSL:
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Re: qPCR of environmental data

Postby MultiDAdmin » Tue Feb 03, 2015 4:07 pm

Hi

Please have a look at our example file "C:\Program Files\Multid\Genex6\GenEx demo\Data pre-processing\Interplate calibration.mdf" to better understand the format.

In general you need to:

- Copy the IPC values to a separate column (IPC_assay)
- Create a column that identify the plates (#plate)
- Create a column that identify the IPCs (#IPC)

The following Tutorial is also available:

http://www.multid.se/viewlets/genex/int ... libration/

BR
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