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Normalization of qpcr data


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#1 geetika

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Posted 01 August 2009 - 11:21 AM

Hi,

I am checking expression of certain genes by qpcr (SYBRGreen), i m bit confused how to normalize my data with internal control. I have data for internal control which is quite uniform but i really don't know how to use it for normalization. Is there any free software or files where we can input our data and it will calculate the fold difference?? if anybody has such excel file plz can you email at trivedigeetika@gmail.com

#2 chrisbelle

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Posted 02 August 2009 - 08:26 PM

Hi,

I am checking expression of certain genes by qpcr (SYBRGreen), i m bit confused how to normalize my data with internal control. I have data for internal control which is quite uniform but i really don't know how to use it for normalization. Is there any free software or files where we can input our data and it will calculate the fold difference?? if anybody has such excel file plz can you email at trivedigeetika@gmail.com


how many genes are you studying? what method of relative quantification are you doing? If you are using standard curves or DDCT you can input into excel and give it a formula command to calculate for you. If you use Pfaffl's you can download the REST software. But if you are only studying a few genes, then it's easier to do it manually. you internal control is a housekeeping gene, i presume...

basically in DDCt,
[2 to the power of (target gene control-target gene treated)]/[2 to the power of (reference gene control-reference gene treated)]

will give you your fold change ratio.
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