RNA Isolation from QIAzol sample with miRNeasy Mini Kit v1

__________________________________________________________________________ Purpose This SOP describes the procedure of total RNAextraction from cell and tissue samples using the Qiagen miRNeasy Mini kit and Ambion Turbo DNA-free kit. The miRNeasy Mini Kit combines phenol/guanidine-based lysis of samples and silica–membrane-based purification of total RNA. QIAzol Lysis Reagent, included in the kit, is a monophasic solution of phenol and guanidine thiocyanate designed to facilitate the lysis of tissues, inhibit RNases, and remove most of the cellular DNA and proteins from the lysate by organic extraction. Cells or tissue samples are homogenized in the QIAzol Lysis reagent. After adding chloroform, centrifugation separates the homogenate into aqueous and organic phases. 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 RNeasy Mini spin column, where the total RNA binds to the membrane, and phenol and other contaminants are efficiently washed away. High-quality RNA is then eluted in RNase-free water. Eluted RNA is treated with an Ambion TURBO DNA-free kit to remove contaminating DNA. A final cleanup step by Ethanol precipitation removes traces of DNase buffer and enzyme to avoid effects on downstream applications, like reverse transcription of RNA into cDNA. This protocol extracts the following classes of RNA: total RNA (including mRNA, miRNA, rRNA, tRNA, snRNA, snoRNA, piRNA and lncRNA)