Possible utilization of in vitro synthesized mRNAs specifically expressed in certain tissues as standards for quantitative evaluation of the results of microarray analysis

• Published in: Journal of biochemical and biophysical methods 2007-08, Vol.70 (5), p.755-760
• Main Authors: Kakuhata, Rei, Watanabe, Masahiro, Yamamoto, Takenori, Akamine, Rie, Yamazaki, Naoshi, Kataoka, Masatoshi, Fukuoka, Satoshi, Ishikawa, Mitsuru, Ooie, Toshihiko, Baba, Yoshinobu, Hori, Tomoshige, Shinohara, Yasuo
• Format: Article
• Language: English
• Subjects: Animals; Base Sequence; Calibration; Carnitine O-Palmitoyltransferase - genetics; DNA Primers - genetics; DNA, Complementary - genetics; Fatty Acid-Binding Proteins - genetics; Gene Expression; In Vitro Techniques; Ion Channels - genetics; Microarray analysis; Mitochondrial Proteins - genetics; Oligonucleotide Array Sequence Analysis - methods; Oligonucleotide Array Sequence Analysis - standards; Quantification; Rats; RNA, Messenger - chemical synthesis; RNA, Messenger - genetics; RNA, Messenger - standards; Standardization; Synthesized RNA; Tissue Distribution; Transcription, Genetic; Uncoupling Protein 1
• ISSN: 0165-022X; 1872-857X
• DOI: 10.1016/j.jbbm.2007.04.004

To examine the possible usefulness of in vitro synthesized RNA as standards in microarray analysis, we prepared full-length mRNAs encoded by 3 rat metabolic genes for heart/muscle type carnitine palmitoyltransferase I (M-CPTI), uncoupling protein (UCP1), and heart/muscle type fatty acid-binding protein (H-FABP). Artificial RNA samples were prepared by adding known amounts of these synthetic mRNAs to total RNA from rat liver, and transcript levels of various genes were compared between the prepared artificial RNA samples and total RNA samples of rat liver by using an Agilent oligo microarray system. Upon the addition of these synthetic RNAs, signals from the DNA spots corresponding to these 3 genes were elevated, but those from the DNA spots representing other genes were not markedly influenced. Using the ratio of the increase in signal intensity of DNA spot to the amount of added RNA, we estimated the expression levels of several genes and compared them with the absolute expression levels determined by calibrated Northern analysis. As a result, the absolute transcript levels of 3 genes encoding acidic ribosomal phosphoprotein P0, type-1 voltage-dependent anion channel (VDAC1), and type-2 glucose transporter (GLUT2) were successfully estimated by this procedure. Furthermore, genes specifically expressed in certain tissues such as UCP1 were concluded to be good candidates as standards for use in microarray analysis.