Building the Toolkits to Examine the Expression and Associated Regulation of RBC Alloantigens.

The Rh blood group system is the most polymorphic and clinically significant blood group system after ABO. The RhD protein, in particular, encompasses numerous conformation-dependent epitopes that can elicit alloimmune responses, leading to hemolytic disease of the fetus and newborn or hemolytic transfusion reactions. Despite this, traditional serologic methods for RhD typing lack sensitivity and specificity to detect RhD variants like partial D and DEL phenotypes. This chapter describes molecular methods to characterize RhD mRNA expression and identify transcript variants in immortalized leukemic or erythroid cell lines and primary reticulocytes. Conventional PCR and quantitative real-time PCR assays were developed using primers designed against the full-length transcripts of RhD and RhCE and known splicing isoforms of RhD. The relative abundance of RhD isoforms in K562 cells was determined from public RNA-seq databases such as Cancer Cell Line Encyclopedia to guide primer design and template input amounts. The methods detail RNA extraction, cDNA synthesis, PCR amplification, and quantitative real-time PCR quantification protocols, with representative results shown for multiple cell lines that are widely used in research settings. To enable unbiased discovery of novel RhD transcript isoforms, a Nanopore long-read cDNA sequencing approach was established. As an orthogonal method, the flow cytometry assay was optimized to detect the RhD full-length protein on the cell surface. These molecular and cellular approaches complement serologic methods and support the transition to genomics-based approaches for Rh typing. Transcriptome-level data, supported by protein-level validation, can provide insights into the regulation of RhD expression and the functional impact of variants. These protocols can be readily applied to erythroid cell lines for mechanistic studies and extended to other blood group genes.