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External standard for qRT-PCR


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#1 Chromi22

Chromi22

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Posted 27 June 2010 - 09:43 PM

I have started a new project and one of the techniques I want to use is qRT-PCR.



First of all let me describe my research topic:

The barley yellow dwarf virus (BYDV, ssRNA-virus) and the wheat dwarf virus (WDV, (DNA-virus) are widely distributed viral diseases of cereals. The virus BYDV is transmitted by aphids and WDV is transmitted by the leaf-hopper Psammotettix alienus.

With the qRT-PCR I want to know which aphid or leaf-hopper carries the virus and possibly the number of virus particles.

What do you think is the best strategy: absolute or relative quantification?

In addition, could you possibly refer me to a company who can synthesise an RNA standard for the RNA virus with a length of 100-200bp?

And could anybody of you give me a hint how does a external standard works like in Vermeulen et.al., External oligonucleotide standards enable cross laboratory comparison and exchange of real-time quantitaive PCR data in Nucleic Acids Research, 2009? Is it possible to use a stuffer sequence between every fwd and rev primer of a particular gene and run it as an external standard? :D
Thanks for any advice/help you may be able to provide.

Cheers Nadine




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