College of Life Sciences, Capital Normal University, Beijing 100048, China.
J Genet Genomics. 2010 May;37(5):341-6. doi: 10.1016/S1673-8527(09)60052-7.
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-dUTP. 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 surface. 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.
mRNA 定量在分子生物学研究中非常重要。传统的分光光度法无法区分 mRNA 与 DNA、rRNA 和 tRNA 种类。Northern blot 可用于 mRNA 定量，但众所周知，该方法耗时。为了快速检测 mRNA 水平，我们开发了一种基于光薄膜生物传感器芯片的方法，用于定量样品中的 mRNA。提取总 RNA 后，用 oligo(dT)(20)引物和 dNTP 与地高辛(DIG)-11-dUTP 逆转录带有 poly(A)尾的 mRNA。转录的第一链 cDNA 与点在光薄膜生物传感器芯片上的 oligo(dA)(20)核苷酸探针杂交。通过洗涤去除多余的第一链 cDNA、单链 RNA 和错配的 DNA/DNA 杂交体。用抗-DIG-AP（碱性磷酸酶）缀合物检测完全匹配的 DNA/DNA 杂交体，然后用 NBT/BCIP 底物孵育显色。根据芯片表面沉积的 cDNA 质量，颜色范围从紫红色到蓝色。对拟南芥样品中 mRNA 水平的检测证明，该方法适用于 mRNA 定量，并且在各种生物体的 mRNA 定量中具有很大的应用潜力。
