Detection of cell-free lncRNA in serum of cancer patients

Abstract

The analysis of circulating RNA molecules is of increasing interest since tumor-specific RNA expression patterns could be a useful cancer biomarker. A new entity of RNA molecules, the so-called long non-coding RNAs (lncRNA), are of particular interest because of its high tissue- and tumor-specificity. The importance of analytical factors in the quantification of lncRNAs is largely unclear and should therefore be investigated in the present study. Serum RNA was isolated from patients with bladder, prostate and kidney cancer as well as patients with non-malignant disease. Analytical variables like different RNA isolation procedures, cDNA synthesis and preamplification were studied with respect to quantification of MALAT1 and ACTB via real-time PCR. The quantification of cell-free serum RNA is feasible although the levels of ACTB and MALAT1 were often only slightly above the detection limit. RNA isolation with a combined phenol-based column purification (Ambion mirVana PARIS miRNA Isolation Kit; Qiagen miRNeasy Serum/Plasma Kit) was most effective. The elimination of DNA contamination was most successful during cDNA synthesis with (Takara-Bio PrimeScript RT Reagent Kit with gDNA Eraser). Preamplification with the Applied Biosystems TaqMan PreAmp Master Mix Kit improved sensitivity. Serum ACTB and MALAT1 levels were not significantly increased in patients with urological tumors compared to patients with non-malignant diseases. An optimized protocol for the analysis of circulating lncRNAs is described in the present study.