Protocol Online logo
Top : New Forum Archives (2009-): : PCR, RT-PCR and Real-Time PCR

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.

 

 

-sshhppqq-

cDNA.

 

all data bases for RNA show just cDNA.

-memari-

Hi memari

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.

Mus musculus glyceraldehyde-3-phosphate dehydrogenase (Gapdh), mRNA

1,254 bp linear mRNA  NM_008084.2

Mus musculus glyceraldehyde-3-phosphate dehydrogenase, mRNA (cDNA clone MGC:175697 IMAGE:40131113), complete cds

1,178 bp linear mRNA  BC145810.1

Mus musculus glyceraldehyde-3-phosphate dehydrogenase, mRNA (cDNA clone MGC:175699 IMAGE:40131115), complete cds

1,178 bp linear mRNA  BC145812.1

 

So, the first one is my choice, right?

 
 
 

-sshhppqq-

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:

 

https://en.wikipedia.org/wiki/Glyceraldehyde_3-phosphate_dehydrogenase

 

in this page on the right you can find Refseq for Human

http://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?val=NM_001256799

and mouse:

http://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?val=NM_008084

 

In these two page above, on the right look for "Pick Primers"

Now you go to this page:

http://www.ncbi.nlm.nih.gov/tools/primer-blast/index.cgi

 

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"

-memari-