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housekeeping genes on a different plate

housekeeping genes on a different plate - Real-Time PCR and Quantitative PCR Forum

housekeeping genes on a different plate - Real-Time PCR and Quantitative PCR Forum


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  #1  
Old 06-12-2011, 09:30 PM
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Default housekeeping genes on a different plate



hi
i just came across a paper that links one of my housekeeping genes to my disease which means i can't use that particular one. (double EEK)

Am i screwed? Or can i use the same cDNA to run a plate with another housekeeping gene on it and use that data to normalize my samples. Any help would be much appreciated. Thanks.

(i can't re-run my qPCR plates with the new housekeeping gene.)
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Old 08-03-2011, 11:02 PM
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Default Re: housekeeping genes on a different plate

Depending on how much cDNA you have left, you should run plates with several reference genes. I typically go for 6-10 reference genes and then use software (either GeNorm or BestKeeper) to determine which ones should be used. Then use the geometric mean of your expression normalized to at least 2 genes. However if you only have enough cDNA for one plate, choose a gene and hope there are no significant differences in its expression with or without the disease.
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Old 08-06-2011, 06:48 AM
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Default Re: housekeeping genes on a different plate

Sunglassesatnight is correct. Prior to any relative qPCR gene expression work, you need to identify the most stable reference genes across the tissues tested. you'll need to select several reference genes, run amplification efficiency and melt curve test for specificity of amplified product, then perform qPCR with all the cDNA extracted from tissues of interest. The Cq values obtained can be analyzed using qBase plus software programme which geNorm has been incorporated into qBase plus. The software will indicate which reference genes are most stable across the tissues and how many reference genes are required for normalization. The number of reference genes is dependent on type of tissues tested and the stability of reference genes expression in them. the minimum reference genes required for normalization is 2. you can refer to MIQE guidelines for qPCR.
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Old 08-26-2011, 08:50 PM
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Default Re: housekeeping genes on a different plate

Just to add to JennYeap's comments (which are very informative), if you don't have the spare 600 euros to buy qbase you can always use excel. Refer to Karlen et al (2007), Statistical significance of quantitative PCR in BMC Bioinformatics vol. 8 no. 131. It explains the best formulae for relative quantification. It's just useful if you want to calculate by hand instead of letting a software do it for you.
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Old 01-18-2012, 08:02 PM
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Default Re: housekeeping genes on a different plate

You can also download the free version of GenEx. In addition to geNorm it has also Normfinder.
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Old 06-12-2012, 09:33 AM
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Default Re: housekeeping genes on a different plate

This topic is answering an old question I had.

So, if i understand all you said, it is possible to run all my housekeeping genes on a single plate, and then get the Ct data, and make a mean on it.

Next, I can use theses data for the normalization of all my target genes that I will run later on other PCR plates ? I do not have to run again the reference genes on each plate ?

In fact, we can run the housekeeping genes one single time, and then use the data for all the following PCR.
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  #7  
Old 02-11-2013, 07:11 AM
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  #8  
Old 02-13-2013, 06:42 AM
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Default Re: housekeeping genes on a different plate

Depends on the plae layout you used and if you included IPC. Check:

tataa.com/products-page/quality-control/tataa-interplate-calibrator

if you ref gene was found differentially expressed in a whole transcriptome stud (microarray or NGS) it can be a false positive. Best is to vaidate it yourself. There are several companies offering ready to go panels. The panel from TATAA includes geNorm and Normfinder (within GenEx) so you don't have to buy software for the analysis:

tataa.com/products-page/gene_expression_assays_panels/reference-gene-panel

you find more info on this topic on: qpcrforum.com

Good luck
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Old 02-13-2013, 07:41 AM
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  #10  
Old 02-13-2013, 08:22 AM
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