MULTIPLEX RT-PCR METHOD FOR THE ANALYSIS OF THE EXPRESSION OF HUMAN SIALYLTRANSFERASES - APPLICATION TO BREAST-CANCER CELLS

In many cases of human cancer, the appearance of hypersialylated glyca n structures is related to a precise stage of the disease; this may de pend on altered regulation of one or more sialyltransferases genes. Si nce several distinct sialyltransferase enzymes arising from different unique genes transfer sialic acid residues in the same linkage onto th e same acceptor, it is impossible to precisely determine which enzyme is involved in the observed phenotype based on enzymatic assays. We ha ve developed a very sensitive and highly reproducible multiplex revers e transcriptase-polymerase chain reaction technique in order to monito r the expression of four human sialyltransferases genes ST6Gal I, ST3G al I, ST3Gal III and ST3Gal IV in small cell samples. Multiplex PCR am plification using specific primers for each sialyltransferase and dete ction of amplification products by polyacrylamide gel electrophoresis is a method that is fast and easy to handle and has proven to be usefu l for establishing sialyltransferase patterns of expression in breast immortalized cell line HBL100 as well as in breast cancer cell lines M CF-7/6, MCF-7/AZ and MDA.