Establishment of a new method, transcription–reverse transcription concerted reaction, for detection of plasma hnRNP B1 mRNA, a biomarker of lung cancer

Abstract

Development of an early detection marker is one of the most important strategies for improving overall prognosis in lung cancer patients. We previously reported that hnRNP B1--an RNA binding protein--is overexpressed in lung cancer tissue from the early stage of cancer, and found that hnRNP B1 mRNA is detectable in the plasma of lung cancer patients using real-time RT-PCR. The purpose of this study was to establish a quick and simple method for detecting plasma hnRNP B1mRNA for use in screening for lung cancer. TRC, a homogenous method for fluorescence real-time monitoring of isothermal RNA amplification using intercalation activating fluorescence DNA probe, was used to detect plasma hnRNP B1 mRNA. The detection limit of hnRNP B1 mRNA by TRC using synthetic control RNA or total RNA derived from a lung cancer cell line was 25 or 8.65 x 10(2) copies, respectively. Using total RNA extracted from 600 mul of plasma, we detected hnRNP B1 mRNA in 39.1% (9/23) of lung cancer patients, with levels ranging from 1.9 to 19,045.5 copies/100 ng RNA, and in 5.2% (5/97) of healthy volunteers. Copy numbers were not associated with age, gender, smoking status, or histological type of cancer. TRC could detect 10(3) copies of hnRNP B1 mRNA in 10 min. Detection of plasma hnRNP B1 mRNA by TRC is a quick, easy, and non-invasive method suitable for lung cancer screening.