Comments on: How many reference genes do you use in qPCR? http://www.americanbiotechnologist.com/blog/reference-genes-poll/ Advancing the productivity of American Biotechnologists Fri, 07 Jun 2013 10:39:44 +0000 hourly 1 http://wordpress.org/?v=3.4.2 By: Vasiliy Pavlichenko http://www.americanbiotechnologist.com/blog/reference-genes-poll/comment-page-1/#comment-10851 Vasiliy Pavlichenko Fri, 02 Dec 2011 21:01:16 +0000 http://www.americanbiotechnologist.com/blog/?p=3264#comment-10851 How Do you calculate results after using 2 or more reference genes? What method do you use? I used ddCt before but don't know how to analize data with 2 reference genes. How Do you calculate results after using 2 or more reference genes? What method do you use? I used ddCt before but don’t know how to analize data with 2 reference genes.

]]> By: avi_wener http://www.americanbiotechnologist.com/blog/reference-genes-poll/comment-page-1/#comment-7569 avi_wener Thu, 08 Sep 2011 19:00:47 +0000 http://www.americanbiotechnologist.com/blog/?p=3264#comment-7569 Jo- Thanks for the comments. Great advice. One lesson is true to everything in life...it always "depends!" I'm just floored by how many people do qPCR without even thinking about this question or testing their assumptions or validity of their data. Jo- Thanks for the comments. Great advice. One lesson is true to everything in life…it always “depends!” I’m just floored by how many people do qPCR without even thinking about this question or testing their assumptions or validity of their data.

]]> By: Jo Vandesompele http://www.americanbiotechnologist.com/blog/reference-genes-poll/comment-page-1/#comment-7568 Jo Vandesompele Thu, 08 Sep 2011 18:52:29 +0000 http://www.americanbiotechnologist.com/blog/?p=3264#comment-7568 Great question! I answered 2-3 reference genes as we most often use 3, but the correct answer would be "it depends". The more variability in your experiment, the more reference genes you need. Two genes is an absolute minimum number in order to determine their expression stability. Sometimes, this is sufficient to normalize the experimentally induced variation away; sometimes you need more genes. I highly recommend to set up a pilot experiment in which you determine which and how many genes are required for accurate normalization. genorm is a great tool to help you with that process (http://www.genorm.info). Jo Vandesompele Ghent University, professor Biogazelle, co-founder Great question! I answered 2-3 reference genes as we most often use 3, but the correct answer would be “it depends”. The more variability in your experiment, the more reference genes you need. Two genes is an absolute minimum number in order to determine their expression stability. Sometimes, this is sufficient to normalize the experimentally induced variation away; sometimes you need more genes. I highly recommend to set up a pilot experiment in which you determine which and how many genes are required for accurate normalization. genorm is a great tool to help you with that process (http://www.genorm.info).

Jo Vandesompele
Ghent University, professor
Biogazelle, co-founder

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