Abstract
Abstract We present here a novel method for in-situ hybridization (ISH) detection of multiple RNA targets in single cells using standard flow cytometer. This method is based on the use of oligo pairs as probes and bDNA signal amplification for highly sensitive and specific ISH detection of RNA. The oligo pairs are designed to hybridize to specific target sequences and can be readily synthesized. The oligo probes/RNA target complex is detected after signal is generated by bDNA signal amplification. The method is compatible with immunostaining, so simultaneous staining of protein and RNA in single cells is feasible. We demonstrated the co-staining of RNA and surface marker proteins in PBMC, B cells, T cells and leukemic cell lines of U937, K562, Jurkat and M1. In addition, the flow cytometric analysis of cytokine gene induction by lipopolysaccharides/R848 in PBMC was shown by staining with antibody for CD14 protein and probes for RNAs encoding interleukin-1 beta, -6, -8 and tumor necrosis factor-alpha. Taken together, this novel RNA ISH flow cytometry assay offers an invaluable tool for studying immune response, stem cell biology and infectious diseases.
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