What to choose as a calibrator? - When looking at basal gene expression (Rt-qPCR)
Hi Meriem,
following the ddCT method, you'll need calibrators as one or more housekeeping genes to ensure a basal and stable expression line, and samples you'll take hoping the average won't move in contrast to the phenotype you're studying. follow the method at Article Analysis of Relative Gene Expression Data using Real-Time Qu...
fred
Hi,
I think you can get some very useful information from these threads;
https://www.researchgate.net/post/How_to_determine_relative_expression_of_genes_compared_to_a_reference_gene_using_qPCR_Ct_values
and
https://www.researchgate.net/post/How_can_I_determine_the_calibrator_for_RT-PCR_data_analysis
and
https://www.researchgate.net/post/How_can_I_calculate_the_expression_of_gene_using_2-ddct_method
Hope this helps!
To have a more bias-free reference you might use two or three reference (aka housekeeping) genes. I would recommend you to read about the standard curve method, we've just published a web tool that you can use with this method to help you analyse the data (Article QuantGenius: Implementation of a decision support system for...
).Also, check MIQE guidelines for reporting (http://clinchem.aaccjnls.org/content/55/4/611.long) that many journals nowadays require you to follow if you want to publish.
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