RNA Extraction Protocol - RiboPure Kit

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
Written on September 28, 2016