Samples are first lysed and homogenized in Lysis Buffer, which immediately inactivates RNases to ensure isolation of intact RNA. After addition of chloroform, the homogenate is separated into aqueous and organic phases by centrifugation. RNA partitions to the upper, aqueous phase while DNA partitions to the interphase and proteins to the lower, organic phase or the interphase. The upper, aqueous phase is collected, and ethanol is added to provide appropriate binding conditions. The sample is then applied to the RNA Spin Column, where the total RNA binds to the membrane, and contaminants are effectively washed away. Finally, high-quality RNA is eluted in Elution Buffer. The purified total RNA is suitable for use in a variety of downstream applications, including: RT-PCR, RT-qPCR, Northern blotting, nuclease protection assays, cDNA library preparation after poly (A)+ selection, and so on.