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.

#RNAzol Extraction Reagent (RNAzol RT, Molecular Research Center, Catalog # RN 190)

• RNAzol Manufacturer’s Instructions
• “RNAzol RT is used to isolate RNA from tissues, cells, liquid samples or blood. One milliliter is sufficient to process up to 100 mg tissue yielding 50 – 700 μg of large RNA (>200 bases) and 8 – 120μg of small RNA (200 – 10 bases).”

#Direct-zol RNA MiniPrep (Direct-zol RNA MiniPrep, Zymo Research, Catalog # R2050)

• Direct-zol Manufacturer’s Instructions
• “Quick, spin column purification of high-quality (DNA-free) total RNA directly from TRIzol®, TRI Reagent® and other acid-guanidinium-phenol based reagents (RNAzol®, QIAzol®, TriPureTM, TriSureTM, etc.)”

Protocol for Isolation of Total RNA

RNAzol Steps

• Add 1ml of RNAzol RT to no more than 100mg of tissue in sterile RNAse DNAse free microfuge tube
• Homogenize tissue and RNAzol mix with teflon microfuge pestle
• Add 0.4ml of Nuclease Free water per 1ml of RNAzol
• Shake vigorously for 15sec and let sit at room temperature for 5-15min (e.g., for 100mg/ml use 15min)
• Centrifuge samples at 12,000g for 15min
• Remove supernatant into a new RNAse DNAse free microfuge tube

Direct-zol RNA MiniPrep Steps

• Add an equal volume of 96-100% ethanol to the supernatant from the step above and mix thoroughly
• Transfer mixture to Zymo-Spin IIC Column in collection tube 0.7ml at a time
• Centrifuge at 12,000g for 30sec
• Discard flow through and repeat additions and centrifugation as necessary for full mixture
• Add 0.4ml RNA Wash Buffer to the column
• 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)

• Incubate at room temperature for 15min

• Add 0.4ml RNA PREWash to the column
• Centrifuge at 12,000g for 30sec and discard flow through
• Add 0.4ml RNA PREWash to the column
• Centrifuge at 12,000g for 30sec and discard flow through
• Add 0.7ml RNA Wash Buffer to the column
• Centrifuge at 12,000g for 2min and discard flow through
• Transfer the column to a new RNAse DNAse free microfuge tube
• Elute RNA by adding 50µl of DNAse/RNase-Free water directly to the column
• Centrifuge at 12,000g for 30sec
• Aliquot RNA into 4 tubes (3µl for Bioanalyzer, 3µl for Qubit, 2x ~20µl for downstream applications