3.1. RNA Preparation

Isolate, Quantify and Perform QC of Total RNA

We recommend using the QIAGEN AllPrep DNA/RNA Micro Kit (Cat.# 80284) for total RNA isolation from cells or tissues. For FFPE tissue, we recommend Thermo Fisher’s RecoverAll™ Total Nucleic Acid Isolation Kit for FFPE (Cat.# AM1975).

Extracted RNA should be DNA-free. With the QIAGEN AllPrep DNA/RNA Micro Kit, a separate DNase treatment is not necessary since the extract is passed through a genomic DNA eliminator column. If using the RecoverAll Isolation Kit for FFPE tissue samples or another Total RNA isolation method, a separate DNase treatment of RNA samples is strongly recommended before starting the procedure. Follow the DNase treatment instructions in the manufacturer’s RNA isolation kit manual.

NOTE: For small amounts of total RNA where a standard DNase treatment and purification may result in significant sample loss, we recommend using double-stranded DNAse (ArcticZymes, PCR Decontamination Kit, Cat.# 80400-100) following the manufacturer’s protocol.

Quantify total RNA with the Thermo Fisher NanoDrop (or equivalent), and confirm the integrity of RNA in each sample prior to starting the assay by one of the following methods:

• Determine the RIN number using the Agilent Bioanalyzer and Agilent RNA 6000 Pico Kit (Cat.# 5067-1511)
• Determine the RIN number using the Agilent Fragment Analyzer and High Sensitivity RNA Analysis Kit (Cat.# DNF-472-1000)
• Using a gel imager, calculate the 28S:18S rRNA ratio after running the RNA samples on an agarose gel.
  

The RIN number for your RNA samples should be no less than 5. If you are using FFPE RNA samples, you should additionally check the samples to ensure that a significant level (at least 50%) of RNA fragments are larger than 300 nt.