An optimised protocol for the extraction of non-viral mRNA from human plasma frozen for three years.

AIMS To detect non-viral mRNA in human plasma that has been frozen for three years using a new protocol. METHODS Plasma from 15 patients with colorectal cancer and 10 normal subjects was separated and frozen with Trizol at -80 degrees C for three years. As a control measure, plasma from 10 of the 15 patients was separated using the same protocol but no Trizol during storage. After three years, all samples were extracted using Trizol and RNeasy before the reverse transcriptase polymerase chain reaction was performed to detect non-viral beta catenin mRNA. In addition, extraction of three plasma samples by Trizol or RNeasy independently was carried out for comparison. RESULTS beta Catenin mRNA was detected in all 15 patient plasma samples and only one of the 10 normal subjects. In contrast, no beta catenin mRNA was found in the control and patient samples that were independently extracted by Trizol and RNeasy kit. CONCLUSIONS This new protocol is a reliable method for extracting non-viral mRNA from the plasma of patients with cancer after longterm storage for three years. Extractions using Trizol and RNeasy kits independently could not isolate mRNA with sufficient quantity and quality for detection.