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Transcriptome sequencing with pyro


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#1 Mycopath

Mycopath

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Posted 26 May 2010 - 06:19 AM

Hi,

I am about to start a new project where I collect total RNA from different fungal tissues and compare the expression ratio between the two present nuclei of the individual.

I have seven different markers that are specific for the different nuclei (i.e. distinguishes between the nuclei) and with pyrosequencing I can quantify the ratio of present nuclei in diploid/heterokaryotic individuals. The next step is to relate the physical nuclei ratio to gene expression of the same seven genes.

My plan is to do cDNA synthesis from total RNA and to use the cDNA in PCR followed by quantification by using pyrosequencing.

So my question is whether I should use oligo(dT)-primers or random hexamers in the cDNA synthesis?

Other suggestions or ideas are of course welcome.

All the best,

/M




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