Protocol for the extraction of total RNA for use in QPCR and Next Generation Sequencing. Yeilds total RNA that has been DNAse treated and is ready for downstream applications.

#RiboPure RNA Purification Kit (RiboPure Kit, ThermoFisher, Catalog # AM1924)

• RiboPure Manufacturer’s Instructions

• “The Ambion RiboPure Kit is designed for rapid purification of high-quality RNA from tissue samples or cultured cells. The kit combines TRI Reagent with glass fiber filter purification to yield exceptionally pure RNA, free of residual proteins and lipids.”

• used DNase I from DirectZol kit

Protocol for Isolation of Total RNA

Homogenization and Phase Separation Steps

• Add 1ml of TRI Reagent for 50-100mg of tissue in sterile RNAse DNAse free microfuge tube
• Homogenize tissue and TRI Reagent mix with teflon microfuge pestle
• Add 0.2ml of chloroform per 1ml of RNAzol
• Vortex for 15sec and let sit at room temperature for 5min
• Centrifuge samples at 12,000g for 10min at 4°C
  • Centrifugation at temperatures >8°C may cause some DNA to partition in the aqueous phase
• Remove supernatant into a new RNAse DNAse free microfuge tube

Filter Binding, Washing, and DNase Treatment

• Add half the volume of 100% ethanol to the supernatant from the step above and mix thoroughly (e.g. 400µl of supernatant plus 200µl of EtOH)
• Vortex immediately to avoid RNA precipitation
• Transfer mixture to Filter Cartrigde in collection tube
• Centrifuge at 12,000g for 30sec
• Discard flow through
• Add 0.5ml Wash Solution to the column (add EtOH to solution concentration prior to use)
• Centrifuge at 12,000g for 30sec and discard flow through

On Column DNAse Treatment

• Mix 75µl of DNA Digestion Buffer with 5µl of DNase I (6U/µl) from Directzol RNA mini prep

• Incubate at room temperature for 15min

• Add 0.5ml Wash Solution to the column
• Centrifuge at 12,000g for 30sec and discard flow through
• Centrifuge at 12,000g for 30sec to dry column
• Transfer the column to a new RNAse DNAse free microfuge tube
• Elute RNA by adding up to 100µl of DNAse/RNase-Free water directly to the column
• Centrifuge at 12,000g for 30sec
• Aliquot RNA into multiple tubes for downstream applications