5.2. RNA Preparation Protocol

For larger bulk tissue, blood, or cell samples as shown in the Table above, follow the guidelines below to isolate, quantify, and QC total RNA. Generally, deep, quantitative AIR profiling of the most abundant clonotypes (with more than 3 mRNA/UMI copies of CDR-specific mRNA per sample) requires using 50-100 ng of total RNA isolated from whole blood (preferred choice) or PBMC samples.

1. Use the appropriate method to isolate total RNA. We recommend the following RNA isolation kits.
    
   • Whole blood: we recommend using the Tempus (Thermo-Fisher) or PAXgene (QIAGEN) for total RNA isolation. Based on our experience, the collection of 3 ml of whole blood in Tempus RNA Blood Tubes followed by RNA isolation using Tempus Spin RNA Isolation kit provides the most simple and reliable protocol for purification of RNA from whole blood.
   • PBMC, buffy coat, or fresh/frozen tissue: for more than 50,000 cells we recommend using the QIAGEN RNAeasy Micro/Mini Plus Kit for total RNA isolation.
   • FFPE tissue: we recommend using the Thermo Fisher’s RecoverAll™ Total Nucleic Acid Isolation Kit for FFPE (Cat.# AM1975). With FFPE, the yield and quality of isolated RNA can vary significantly, depending on the age and method used to fix the sample. It is critical to assess the quality of the RNA before proceeding with the DriverMap protocol.

2. Ensure the extracted RNA is free of almost all DNA.
    
   • Small amounts of DNA (i.e, <5% genomic DNA contamination) do not interfere with the assay. With the Tempus and RNeasy Plus RNA Kits, a separate DNase treatment is not necessary. When using other RNA isolation protocols, a separate DNase treatment of RNA samples is recommended before starting the procedure. Follow the DNase treatment instructions in the manufacturer’s RNA isolation kit manual.

3. 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 is larger than 300 nt.