RT-PCR quantitation of cytokine responses in vivo from specimens containing small numbers of cells during bioimmunotherapy

A Standard Template method has been developed to carry out semiquantitative RT-PCR analysis on mRNA extracted from small specimens that contain low yields of RNA. Using easily prepared templates (made from previously tested reference specimens), standard calibration curves were generated for each of two cytokine products of interest, specifically IL-10 and IFN-γ. Also, internal standardization was accomplished by quantitating a well-characterized housekeeping gene (GAPDH). Simple densitometry of the RT- PCR product did not demonstrate sufficient reliability for quantitation since a logarithmic relationship was shown between product and template input. Peritoneal exudate cell specimens that were obtained from ovarian cancer patients during intraperitoneal immunotherapy were utilized for the demonstration of IL-10 and IFN-γ transcript in vivo. Briefly, this method consists of: (1) template preparation: a PCR product for the cytokine of interest is generated from a previously identified positive specimen, purified and stored at -20°C. (2) RT-PCR: cDNAs are produced from RNA extracted from patient specimens. Replicates of each cDNA and serial dilutions of the corresponding template are amplified with primers specific for each cytokine of interest. (3) Quantitation: photographs are made of the PCR products displayed on an agarose gel and are analyzed by densitometry. Determinations of fold-differences in cytokine transcript expression are normalized to the mRNA content of each specimen (based on the expression of GAPDH).