I would like to analyse the expression of a number of cytokines in in vitro cultures of dendritic cells and was advised to do this by PCR.
As primer pairs are available, I think that might be a suitable method (albeit more difficult to quantitate than ELISA-based cytokine measurements). However, I did my last PCR about 3 years ago and would be really thankful for a useful protocol for cDNA preparation, RNA isolation and the PCR itself. Do I actually have to use RT-PCR?

I'm extremely grateful for any hint or advice and thank you all in advance!

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Be careful with PCR. mRNA doesn't mean that the cytokine will be synthesized.
you can do PCR for a first screening, but then you should confirm with ELISA.

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that's what I was planning - doing the PCR first to confirm the presence of selected cytokines and do an ELISa afterwards for the PCR-positive cytokines.

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sorry for the protocol, I'm even worse than you, I did few 5 years ago.
I'm sure someone else will give you a good protocol

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hi,

maybe you will find some informations in these 2 papers:

1. Overbergh, L., Valckx, D., Waer, M., and Mathieu, C. (1999) Quantification of murine cytokine mRNAs using real time quantitative reverse transcriptase PCR, Cytokine 11, 305-312.
2. Overbergh, L., Giulietti, A., Valckx, D., Decallonne, R., Bouillon, R., and Mathieu, C. (2003) The use of real-time reverse transcriptase PCR for the quantification of cytokine gene expression, J Biomol Tech 14, 33-43.

Sebastien_

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