After tissue homogenization, the Covance Genomics Laboratory (CGL) extracts total RNA, DNA, and microRNA, from submitted samples, with most sample types running under a high-throughput, automated process. CGL has developed extraction techniques for a variety of biological sources within several species and states of submission.
Chloroform Extraction and Volume Estimation of Unpurified Total RNA
Unpurified total RNA is extracted with a chloroform procedure. Then, its mass used to estimate the volume of the aqueous supernatant recovered. This value determines how much of the sample will be added to the purification columns, where the RNA is captured prior to quality control of the sample (called Sample QC).
The unpurified sample is arrayed into a 96-well plate, where total RNA from individual tissue samples is purified and isolated. The isolation plates are loaded into Biomex FX. The RNA of the sample preferentially binds to the silica-based column and any proteins or cellular DNA impurities are washed away.
After the tissue homogenization and RNA extraction process, samples are stored and controlled in a freezer system until required for further processing.
Total RNA purified samples are tested with a series of parameters to measure quality and quantity. For example, the RiboGreen assay and Agilent Bioanalyzer traces are performed on incoming samples to determine quality and RNA concentration.