Analysis of Endogenously Expressed Double-Stranded RNAs in Human Cultured Cells.

Long double-stranded RNAs (dsRNAs) play a critical role in modulating innate immune responses. In particular, endogenously generated dsRNAs are increasingly recognized as a biomarker and effector in immune-related diseases such as cancer, viral infection, and autoimmune disorders. However, accurate detection of endogenous dsRNAs is challenging due to their transient nature and low abundance. This chapter outlines three complementary methods using the dsRNA-specific J2 antibody: J2 dot blot for semiquantitative assessment of dsRNA levels; J2 immunocytochemistry for visualization of dsRNA subcellular localization; and J2 formaldehyde-mediated crosslinking immunoprecipitation coupled with RT-qPCR and/or high-throughput sequencing for quantification and/or identification of dsRNA species. Together, these methods enable sensitive and reproducible analysis of dynamic dsRNA landscapes under pathophysiological conditions, providing a versatile workflow for studying dsRNA signaling in mammalian cell biology and human disease models.