Molecular methods for analysis and expression of transcobalamin II

Abstract

Publisher Summary Transcobalamin II (TCII) has been reported to be expressed in several mammalian tissues, including liver, kidney, intestinal epithelium, cultured fibroblasts, and human umbilical vein endothelial cells. The expression of TCII has been determined by direct assay for apo-TCII and holo-TCII and by analysis for the TCII transcript in cellular RNA. This chapter discusses the molecular methods for analysis and expression of TCII and uses ribonuclease (RNase) protection and reverse transcriptase-polymerase chain reaction (RT-PCR). The RNase protection requires the synthesis of a radiolabeled TCII riboprobe, incubation of this probe with the messenger RNA (mRNA) prepared from the tissues or cells to be analyzed, followed by RNase A and RNase T digestion. The RNase(s) will digest the regions of the riboprobe that do not form hybrids with the complementary regions of the TCII mRNA. Only the RNA–RNA hybrids will remain intact and can be identified by polyacrylamide gel electrophoresis and autoradiography. The size of the TCII transcript in the RNA preparation can be determined by RT-PCR. A complementary DNA (cDNA) copy is generated from total or poly(A) + RNA by reverse transcription, using an oligo(dT) primer. The TCII cDNA is then amplified by PCR, using specific primers to the extremities of the coding region of the TCII cDNA.