Application of laser-induced fluorescence for quantitative analysis of messenger RNA

从哺乳期小鼠乳腺中提取酪蛋白mRNA和竞争性逆转录/聚合酶链反应（RT-PCR） mRNA。在酪蛋白mRNA中，将分子量为22000的酪蛋白mRNA转录为cDNA并克隆。测定酪蛋白cDNA的核酸序列。将已知外源基因的DNA片段插入酪蛋白cDNA中。在聚合酶链反应中，该同源DNA被用作竞争DNA。采用RT-PCR法测定泌乳期乳腺中酪蛋白mRNA的含量。其数量在泌乳早期较少，在泌乳中期达到最大值，并在泌乳后期逐渐下降。与哺乳期中期母鼠分离后，乳腺中酪蛋白mRNA在分离后9h开始消失。饲喂幼崽后，酪蛋白mRNA升高并恢复到正常水平。首先，用荧光标记的UTP进行RT-PCR扩增酪蛋白cDNA。合并效率很差。其次，将长度为400bp的酪蛋白DNA片段插入质粒DNA；用Spe I酶切DNA后，分别用T7和SP6聚合酶合成荧光标记的正义和反义RNA探针。我们检测到荧光标记RNA探针的量大于10^<-15> mol，可用于Northern blot分析和毛细管电泳。

Cloning of casein mRNA and competitive reverse-transcription/polymerase chain reaction (RT-PCR) mRAN was extracted from lactating mouse mammary gland. Among casein mRNAs, mRNA for casein with the molecular weight of 22,000 was transcribed to cDNA and cloned. The nucleic acid sequence of casein cDNA was determined. DNA fragment of known foreign gene was inserted into casein cDNA.This kimeric DNA was used as competitor DNA during the polymerase chain reaction.Animals and assay conditions The quantity of casein mRNA in the mammary gland was determined by the RT-PCR throughout lactation. Its quantity was small at early lactation, reached maximum at midlactation, and declined gradually toward to the end of lactation. By the removal of pups from the midlactating mother, casein mRNA began to disappear from the mammary gland at 9h after the separation. By supplying the foser pups, casein mRNA increased and returned to the normal level.Fluorescene-labelled probe First, casein cDNA was amplified with fluorescene-labelled UTP by the RT-PCR.The incorporation efficiency was very poor. Secondly, the casein DNA fragment of 400 bp in lenght was inserted into the plasmid DNA.After the digestion of the DNA with Spe I,the fluorescene-labelled sense and antisense RNA probes were synthesized using T7 and SP6 polymerases, respectively. We detected the fluorescene-labelled RNA probe with the amount of larger than 10^<-15> mol and could be used for tne Northern blot analysis and for capillary clectrophoresis.