RNA standards for quantitative PCR

Adrian Allan aka at rri.sari.ac.uk
Mon Feb 3 08:57:03 EST 1997


Beisvåg Vidar wrote:
> 
> Hi, Netters:
> 
> I am trying to set up a quantitative PCR assay. I want to use an internal
> RNA standard. This could be made by in vitro transkription of a cloned PCR
> product. My problem is that i want a poly A tail on the 5`of the finaly
> transkript (and I dont know how I could make such a product). Then I could
> add the RNA standard in the mRNA isolating step. I want the standard to
> compeet with the mRNA about the olido dT beeds, who I am using for
> isolation of mRNA. If I can do this, it hopefully will be a good assay for
> quantification of expressed genes with help of PCR.
> 
> I hope some one could help me, or know some one who could give me a hint!
> 
> Thanks
> 
> Vidar

Vidar

If you are intent on using magnetic beads for mRNA isolation I suggest 
you read this paper:

Competitive PCR for quantitation of gonadotrophin-releasing hormone mRNA
level in a single micropunch of the rat preoptic area, K. Kim et al,
Molecular and Cellular Endocrinology, 97, (1993) 153-158


In the paper, the mRNA is isolated from the sample using Dynabeads mRNA 
purification kit. When the oligo dT beads are bound to the mRNA, the mRNA 
is then eluted off the beads with the kit's elution buffer (2mM EDTA). 
The RNA standard is then added to the eluted mRNA and both are reverse 
transcribed with random hexamers, and the quantitative RT-PCR can be 
continued.

If you wanted to have a standard with a poly-A tail I believe there was a 
paper in Biotechniques about two years ago which used a modified 
antisense primer with a string of dT's, so when PCR was done to make the 
standard the product had a poly A tail at the 3' end. I guess you could 
purify the standard using oligo dT beads and then elute it off. I'll dig 
out the reference if you need it.
 
Alternatively, you can forget about using beads, and just do a total RNA 
isolation and reverse-transcibe both RNA's using random hexamers, or one 
or both of your PCR primers. It's up to you. 

Good Luck,

Adrian Allan



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