Extraction and qPCR-Based Detection of miRNAs from Cultured PBMCs of Bubaline Origin
MicroRNAs are small noncoding but functionally important RNA molecules that are involved in regulating diverse cellular, metabolic, and immune processes. Their small size necessitates modification in traditional acid phenol-chloroform based RNA isolation procedures to get highly enriched fraction of small RNA that includes miRNAs and siRNAs. Further, of the different methods available, real-time PCR is a powerful tool for precise and specific detection and quantification of miRNA. Moreover, real-time PCR is used to validate the screening or expression of miRNAs that are discovered during high-throughput sequencing, or microarray analysis. We demonstrate here the method of extraction of miRNAs from cultured PBMCs of bubaline origin followed by the qPCR-based (both SYBR green and TaqMan-based chemistries) identification of miRNAs expressed in response to TLR ligand stimulation.
Key wordsSmall RNAs PBMCs TaqMan MicroRNA assay miScript primer assay
The work was supported by SERB-DST (Ministry of Science of Technology, Government of India) sponsored Research Grant SERC-LS-293-2011 (File Number SR/FT/LS-22/2011).
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