Quantitative measurement of protein S gene expression by reverse transcription‐competitive PCR method; determination of mRNA level in fractionated human peripheral blood cells and various cultured cell lines

Abstract

We developed the measurement system for human protein S mRNA (cDNA) by a reverse transcription-competitive polymerase chain reaction (RT-competitive PCR) method with a compact digital camera. The sensitivity of the method was 100,000 fold more sensitive than the traditional northern blot analysis. We confirmed the accuracy and usefulness of the system by measuring the absolute values of protein S mRNA in fractionated peripheral blood cells, namely platelets, mononuclear cells, granulocytes and red blood cell from healthy human and six different cultured disease cell lines tested, namely, lung carcinoma, stomach carcinoma, hepatoma, highly differentiated hepatoma, promyelocytic leukemia and monocytic leukemia. The amount of protein S mRNA in the platelet rich fraction (6.89 x 10-19 mol /ptg total RNA) was the largest of all fractions tested and that in the mononuclear cells was the second largest. In the fractions of granulocytes and red blood cells, no protein S mRNA was detected. The detected values in five cell lines ranged from approximately 5.6 x 10 -19 to 97.2 x 10 -19 mol / pg total RNA. The content was reduced in the order, highly differentiated hepatoma > hepatoma > lung carcinoma > stomach carcinoma > monocytic leukemia. Promyelocytic leukemia cell line had no expressed protein S mRNA. Thus, the assay system described here was sensitive, reproducible, simple and useful for the quantitative measurement of an infinitesimal level of protein S mRNA to characterize normal and/or patho-physiological states of human blood and somatic cells.