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TaqMan gene expression assays - (Dec/10/2014 )

Dear All,

 

I have some questions regarding cDNA synthesis and qPCR - hope you can help me:

I am using High Capacity cDNA RT kit from Life technologies, and then I will be using TaqMan Gene Expression Assay.

My questions are:

1) How much should I use of my RNA (concentration) while performing cDNA synthesis?

2) How much should I dilute my cDNA while performing the RT-PCR?

3) Is it required to DNase treat my samples (human tissue)?

 

Yes, I am aware of that I should do some pilot experiments, but I cant because limitations of the samples. 

 

I hope you will help me - I am really new to this field.

 

Thank you.

 

Chris

-chrishansen-

Hi,

 

1) Up to 1 ug per 10 ul. I would start with total 500 ng per 10 ul of RT reaction volume. If you have really small amount of RNA, try 300 ng per 10 ul.

2) Depends on what you are looking at with respect to expression level. I dilute 1:10, but you may dilute less (e.g. 1;5) depending on how much sample you have to use.

3) In principle yes, but some DNAses also diminish the levels of RNA. As minimum you should control for "no reverse transcriptase" reaction to see the presence of genomic DNA in your samples. In addition, check if your taqman probes (including your housekeeping normalizers) span exon-exon junctions , and in this case you should not amplify genomic DNA (in principle). 

-Janne-