Description:
The miEASY Mini Kit combines phenol/guanidine-based lysis of samples and silicamembrane based purification of total RNA. Lysis/Binding buffer is a monophasic solution of phenol and guanidine thiocyanate, designed to facilitate lysis of tissues, to inhibit RNases, and also to remove most of the cellular DNA and proteins from the lysate by organic extraction.
Cells or tissue samples are homogenized in Lysis/Binding buffer. 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 extracted, and ethanol is added to provide appropriate binding conditions for all RNA molecules from 18 nucleotides (nt) upwards. The sample is then applied to the RNA Bind columns, where the total RNA binds to the membrane and phenol and other contaminants are efficiently washed away. Highquality RNA is then eluted in RNase-free water.
For enrichment of miRNAs and other small RNAs (less than ~200 nt) in a separate fraction, a specialized protocol is provided in Appendix A, page 31. Enrichment of small RNAs in a separate fraction may be advantageous for certain applications where mRNA and rRNA could lead to increased background. For this specialized protocol, an additional microRNA Bind Columns is inclued.
