Question about the RT PCR - (Dec/19/2013 )
Hi All experts.
I have some questions about RT-PCR.
I want to examine the gene expression level after drug application.
I use RNeasy to get total mRNA and then follows two steps RT-PCR process.
In the following RT-PCR steps, I do the reverse transcript with iscript cDNA synthesis to obtain the cDNA.
Now. my problem is the primer design.
What kind of template I need to use? mRNA, mRNA complete cds, DNA
Thank you very much.
all data bases for RNA show just cDNA.
Thank for your response.
What does it mean? "all data bases for RNA show just cDNA". Could you help me explain it more clearly?
Take GAPDH as example, I found these.
1,254 bp linear mRNA NM_008084.2
1,178 bp linear mRNA BC145810.1
1,178 bp linear mRNA BC145812.1
So, the first one is my choice, right?
There are some sequences for GAPDH in databases.
The most important is RefSeq (mRNA) for GAPDH.
Search GAPDH in google and go to this like for WIKIPEDIA:
in this page on the right you can find Refseq for Human
In these two page above, on the right look for "Pick Primers"
Now you go to this page:
that you can change settings.
Also you can check this item below to desing a pair of primers to amplify all variants of your gene of interest:
"Allow primer to amplify mRNA splice variants"