Chicken RNA Integrity Assessment through Capillary Electrophoresis for miRNA Expression Study using RT-qPCR

Abstract

The main concern in RNA research is the risk of degradation of RNA in the experimental conditions due to the ubiquitous nucleases and detecting the RNA quality through RNA integrity score provides better understanding of degradation process. Conventionally, the total RNA quantity and purity measured using spectrophotometer but even a standard reading stands questionable due to contributions from protein and genomic DNA and doesn’t ensure appropriate discrimination among RNA fractions. In present study, we performed RNA integrity analysis using capillary electrophoresis system for extracts from chicken liver, heart and gizzard. The optical measurement of RNA quality through measurement of A 260/280 ratio stands non reliable since below par RIN (RNA Integrity Number) were reported for both liver and gizzard extracts in capillary electrophoresis whereas heart extracts reported a RIN of 7.6 (optimum is 7). The RIN score of 6.8 for liver and 5.4 for gizzard were well correlated with the degraded size of 28S subunit and 18S subunit of ribosomal RNA respectively. In comparison with conventional method, capillary electrophoresis based RNA quality evaluation provides reliable information and such data can be used in selecting the RNA extracts for miRNA expression study in RT-qPCR.