problem of normalization gene in RTqPCR

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moeez
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problem of normalization gene in RTqPCR

hi
i have a problem in RTqPCR. i tried three different genes of normalization (GAPDH, B-actin and S26) but each time these 3 genes have different values.
i am using stable cell lines (p65,c-Rel and IKB) of jurkat for extraction of RNA.
for making of cDNA i use 1000 ng RNA and use Superscript III reverse transcriptase (invitrogen).
is thre any problem with my RNA samples or stable cell line give different values.
please suggest something for my problem.

Ivan Delgado
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Hi moeez,

Getting different results when using different reference genes is not unusual. GAPDH is actually well known for varying in gene expression across different samples so it is not considered a good reference gene for a number of cell lines. Ribosomal genes like S26 have a very high level of gene expression, which may not be ideal when using it as a reference gene to study the gene expression of genes with lower gene expression. 

My advice is to look for other reference genes. If you find at least two reference genes that give you similar results then you got what you were looking for. Alternatively, if the reference genes you are using show similar levels of gene expression across the samples you are studying, then you can use all three together as internal standards. In other words, you can normalize the expression of your genes using the average of all these three reference genes. This only works if all three of these reference genes show very similar gene expression across your samples. In other words, none of these three reference genes show much difference in gene expression across all your samples. 

Good luck.

moeez
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thanks ivan

thanks ivan
i will try this tomorrow and then let u know;

Biju
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Hi moeez,

Hi moeez,
 I additon to what was written, you should also check the shelf life of the  reference std especially if the std has been opened long time ago. In this case,  possibily exists that exposure to light has caused deterioration of the quality of the Taqman std. If you compare the color  of the std in use with an unopened vial of the same std taken freshly from the -200 freezer, you could see a decreased color for the opened vial. This could result in worsening levels for expression of the reference std over a perid of time(even one week is sufficent some imes) .
Biju Joseph

Strosa8
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I work with 3t3-l1, which we

I work with 3t3-l1, which we differentiate into fat cells.  We have used actin as our housekeeping gene for qRTPCR, but may try and switch to 16sRNA or GADPH.  Although actin levels themselves aren't altered due to our treatments, their levels vary depending on the differentiation stage/age/condition of the cells.  If none of your housekeeping genes are consistent between your samples, you may need to make sure there aren't any other factors (such as cell health, age of cells or treatment effects) that may be influencing the housekeeping gene.  There are usually lots of housekeeping genes to try out, so do some research and find out which ones are most consistent in yor samples.