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 semiquantit ative RT-PCR analysis on mRNA extracted from small specimens that cont ain low yields of RNA. Using easily prepared templates (made from prev iously tested reference specimens), standard calibration curves were g enerated for each of two cytokine products of interest, specifically I L-10 and IFN-gamma. Also, internal standardization was accomplished by quantitating a well-characterized housekeeping gene (GAPDH). Simple d ensitometry of the RT-PCR product did not demonstrate sufficient relia bility for quantitation since a logarithmic relationship was shown bet ween product and template input. Peritoneal exudate cell specimens tha t were obtained from ovarian cancer patients during intraperitoneal im munotherapy were utilized for the demonstration of IL-10 and IFN-gamma transcript in vivo. Briefly, this method consists of: (1) template pr eparation: a PCR product for the cytokine of interest is generated fro m a previously identified positive specimen, purified and stored at - 20 degrees C. (2) RT-PCR: cDNAs are produced from RNA extracted from p atient specimens. Replicates of each cDNA and serial dilutions of the corresponding template are amplified with primers specific for each cy tokine 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 expr ession of GAPDH). (C) 1998 Elsevier Science B.V. All rights reserved.