Isolation of good quality ribonucleic acid (RNA) from mammary glands of elite animals is often hindered by invasive method of mammary tissue sampling, ethical permission, time consuming, and repeated biopsies from the same animal at different time points. The aim of the present study was to optimize a protocol for RNA isolation from milk fat that is suitable for transcriptome analysis. In this study, authors isolated good quality RNA from milk fat of goats and buffalo milk by combining two methods namely Trizol and GenElute mammalian RNA isolation kit. The results showed good quality as well as high quantity of total RNA suitable for downstream applications like reverse transcriptase-quantitative polymerase chain reaction and next generation sequencing.