title: An efficient method for high-fidelity messenger RNA amplification from a small amount of total RNA.
abstract: Comprehensive analyses of gene expression have been carried out by the development of microarrays and deep sequencers. However, it is difficult to obtain comprehensive information on gene expression from a small amount of ribonucleic acid (RNA). Therefore, we investigated the reproducibility and application of T7 RNA polymerase-mediated transcription, adaptor ligation and polymerase chain reaction (PCR) amplification, followed by T7 transcription (TALPAT), an efficient method for amplifying poly (A)-positive RNA, such as messenger RNA (mRNA). When amplified complementary RNA (cRNA) was electrophoresed, a large number of amplified cRNA was detected in the size of 0.2-0.5 kb. This indicates that the region up to 0.2-0.5 kb from the 3' end of the original mRNA was amplified by the TALPAT method. Seven housekeeping genes, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), hydroxymethylbilane synthase (HMBS), hypoxanthine phosphoribosyltransferase (HPRT1), ribosomal protein L13a (RPL13A), succinate dehydrogenase complex (SDHA), TATA box-binding protein (TBP) and ubiquitin C (UBC), showed high reproducibility (square of the correlation coefficient, R2=0.9954), according to scatter plots of Ct values obtained in the real-time PCR analysis of amplified cRNA. In addition, relative expression ratios of amplified cRNA of the seven housekeeping genes were approximately equal to the ratio of the original RNA solution. Furthermore, cRNA was amplified from 20 pg total RNA. In the present study, we confirmed the characteristics of mRNA amplification using the TALPAT method. This method may be applicable to mRNA and poly (A)-positive non-coding RNA amplification, using a small amount of RNA from single, laser-captured and sorted cells, as well as exosomes from serum, urine and body fluids.
authors.affiliation: Department of Biomedical Sciences, Division of Medical Life Sciences; ; Research Center for Biomedical Sciences;
authors.forename: Mitsuru
authors.initials: M
authors.lastname: Chiba
authors.affiliation: Department of Medical Technology, Hirosaki University Graduate School of Health Sciences, Hirosaki, Aomori 036-8564, Japan.
authors.forename: Saya
authors.initials: S
authors.lastname: Asari
authors.affiliation: Department of Medical Technology, Hirosaki University Graduate School of Health Sciences, Hirosaki, Aomori 036-8564, Japan.
authors.forename: Misako
authors.initials: M
authors.lastname: Kimura
authors.affiliation: Department of Biomedical Sciences, Division of Medical Life Sciences; ; Research Center for Biomedical Sciences;
authors.forename: Toshiya
authors.initials: T
authors.lastname: Nakamura
keywords: T7 in vitro transcription; adaptor ligation; complementary ribonucleic acid synthesis; mRNA amplification; real-time polymerase chain reaction