normalization for real-time PCR - (Jan/14/2007 )
We use HPRT as house keeping gene to normalize our result. However there are always 10-fold or higher differences of the amount of HPRT among the samples. (For example, in one sample the HPRT Ct value is 20, and in other one it is 25). Some one told me if there was a difference more that 3-fold of the house-keeping gene, the result should not be used. And he suggested me to use the same amounts of RNA for RT-PCR, but it is impossible for me to calculate the amounts of RNA. So could you guys give me some suggestion? thanks
well you may choose an other house keeping gene, but why a OD measure is impossible for you?
Thanks for ur reply. But i don't think the difference come from the housekeeping gene we choose. it may because the difference of amount of rna we used.
we isolate RNA from urine sample of patient( 100ml). i once tried to measure the OD, but the value was so small. so i cannot calculate the amounts of RNA. then i run a agarose gel, but i can not see any band. it may becasue small amount.
what i want to know is that can i use the reslut if there are 10 times difference amount different samples?
Or do you have any other good way to solve the problem?
Thanks a lot.
10timles difference is out limit.
I mention to use an other housekeeping gene to be more confident of normalization if you have 2-3 times differences between samples
Moreover there may be a possibility that youre housekeeping gene is changing, isn't it?
did you try nanodrop to have an idea of the measure?
How do you extract your RNA more precisely?
Seems to me that 100ml should give enough RNA for quantification...
I once used 18s RNA as housekeeping, but there had the same problem.
There is no nanodrop in our lab. but i will try to find one.
our protocol is as follow:
urine was centrifuged at 2000g for 30min at room temperature.
and the pellet was washed with PBS.
and then centrifuged at 12000g for 15min.
and isolated rna from the cell pellet using RNeasy mini kit (QIAGEN).