Traget gene has the same Ct as HK gene - what to do ? - HELP !!!!!!!!! (Apr/17/2007 )
Hi,
I have a problem... I'm trying to qRT GFAP expression(my HK gene is GAPDH).
both primer sets (GFAP, GAPDH) were calibrated and have good efficiency (slopes are -3.12,-3.2 respectively).
the problem is that both have the same Ct (~17).
What can i do ???
HELP !
Lior.
Well depends on your question, are you comparing treated vs. untreated cells? Then you can use the values. Maybe it is a good idea to use another HK gene?
yes I'm comparing a couple of treatments. I can use the values but what do i use as an internal control ? do u really think a different HK will do the difference ? which one do u recommend ? Bactin ? or something else.
I don't know if it is important but i use SYBR with 10ng of cDNA template.
If you have a deltaCt value for your control experiment that equals zero, you can still calculate deltadeltaCt values! For example, imagine your untreated samples have a deltaCt of 0 and your treated sample has deltaCt of -3. According to the formula:
deltadeltaCt=2(deltaCttreatment-deltaCtcontrol)=2(-3-0)=0.125
So you have an 8fold reduction in the gene's expression.
Okay?
Still, it is recommended to use several HK genes and normalize them, as many HK genes are also differentially regulated (a software that generates a normalized HK gene is for example geNorm)
Thanks guys.
meanwhile i found this table in Qiagen (ndogenous control genes in real-time RT-PCR) with gibes relative expression levels of HK genes in human and mouse. i thonk i will try a diffrernt HK (low expression such as Hprt (Hypoxanthine guanine phosphoribosyl transferase)).
http://www1.qiagen.com/products/pcr/quanti...epingGenes.aspx