Evaluation of a co-extraction kit for mRNA, miRNA and DNA methylation-based body fluid identification.

Abstract

Recently, messenger RNA (mRNA), micro RNA (miRNA), and DNA methylation (DNAm) have been reported as novel markers for body fluid identification (BFID). Comprehensive analysis of these markers should be a flexible and reliable BFID method for various types of forensic samples. However, independent extraction of all targets can be difficult depending on the usable amounts of samples. In this study, the applicability of a co-extraction kit for these molecules, the AllPrep DNA/RNA/miRNA Universal Kit (APU), was evaluated by comparing RNA and DNA extracted from blood and saliva stains by the APU with those extracted by standard kits for each molecule and by previously reported methods for mRNA/DNA or miRNA/DNA co-extraction. Electrophoresis using the Bioanalyzer platform and real-time PCR analysis revealed that the APU performed almost equivalently to each standard kit in the quality of RNA or DNA extracted and extraction efficiency of mRNAs, miRNAs, and DNA. Moreover, the APU outperformed the co-extraction methods, especially in RNA integrity and miRNA extraction efficiency. In addition, pyrosequencing revealed that the methylation ratios of DNA extracted by the APU were not different from those extracted by standard DNA extraction kits. Overall, the APU is applicable to comprehensive analysis of mRNA/miRNA/DNAm markers for BFID analysis. Because the DNA eluate can also be used for DNA typing, the APU may be among the best choices for forensic examination of body fluid samples in terms of its flexibility and reliability in BFID and efficiency in sample consumption.