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The study reports optimization of RNA extraction method from buffalo milk fat, to be used for gene expression analysis. Higher yields were obtained from buffalo milk fat as compared to skim milk, with manual method as well as commercial kits. Quality and integrity of the samples from both methods was comparable. Quantitative profiling of mammary gland specific genes from skim milk and fat derived RNA validated the efficiency of MFG as an alternate source of total RNA. This study confirmed that the RNA obtained from buffalo MFG was suitable for studies of mammary genes expression.