Feasible and reliable quantification of mRNA in Arabidopsis thaliana using optical thin-film biosensor chips

作     者：Sulan Bai Fan Wang Zhen Zhang Shucheng Li Jie Zhang Yaochuan Zhang 

作者机构：College of Life Sciences Capital Normal University Beijing 100048 China Beijing Vocational College of Agriculture Beijing 102442 China 

出 版 物：《Journal of Genetics and Genomics》 (遗传学报（英文版）)

年 卷 期：2010年第37卷第5期

页      面：341-346页

核心收录：

学科分类：0710[理学-生物学] 083002[工学-环境工程] 0830[工学-环境科学与工程（可授工学、理学、农学学位）] 07[理学] 08[工学] 09[农学] 0903[农学-农业资源与环境] 0713[理学-生态学] 

主　　题：mRNA quantification optical thin-film biosensor chip assay 

摘      要：mRNA quantification is very important in molecular biological researches. Traditional spectrophotometric method cannot distinguish DNA, rRNA and tRNA species from mRNA. Northern blot can be used for mRNA quantification but is known to be time consuming. To rapidly detect mRNA levels, we developed an optical thin-film biosensor chip based method, to quantify mRNA in samples. After total RNA was extracted, the mRNA with poly（A） tails was reverse transcribed with oligo（dT）20 primers and dNTPs mixed with digoxigenin（DIG）-11-dUTE The transcribed first strand cDNA was hybridized with oligo（dA）20 nucleotide probes spotted on optical thin-film biosensor chips. Excess first strand cDNA, single-strand RNA, and mis-matched DNA/DNA hybrids were removed by washing. The perfect-matched DNA/DNA hybrid was detected with anti-DIG-AP （alkaline phosphatase） conjugate and then incubated with NBT/BCIP substrate for color development. The range of the color is from purplish red to blue, according to the cDNA mass deposited on chip sur- face. Detection of mRNA levels from Arabidopsis samples proved that this method is feasible for mRNA quantification, and has great potential for application in mRNA quantification in various organisms.