Ponce M R, Pérez-Pérez J M, Piqueras P, Micol J L
División de Genética, Universidad Miguel Hernández, Alicante, Spain.
Planta. 2000 Sep;211(4):606-8. doi: 10.1007/s004250000371.
A non-radioactive, rapid and sensitive method is presented for the simultaneous detection of several mRNA molecules. The technique is based on conventional first-strand cDNA synthesis by reverse transcriptase, followed by multiplex polymerase chain reaction (PCR) co-amplification of several gene products in a reaction mix containing several primer sets, each including a fluorescently labeled oligonucleotide. The PCR products obtained are finally electrophoresed in a single lane of a polyacrylamide gel, in an automated DNA sequencer controlled by fragment-analysis software. The method has proven useful to efficiently detect nine mRNA transcripts, some of which are low copy number, from small specimens such as single flowers and leaves of Arabidopsis thaliana (L.) Heynh. This approach might be easily extended to other biological systems, for developmental and physiological analyses, population studies and diagnosis.
本文介绍了一种用于同时检测多个mRNA分子的非放射性、快速且灵敏的方法。该技术基于通过逆转录酶进行的常规第一链cDNA合成，随后在含有多个引物组的反应混合物中对几种基因产物进行多重聚合酶链反应（PCR）共扩增，每个引物组都包含一个荧光标记的寡核苷酸。最终，将获得的PCR产物在由片段分析软件控制的自动DNA测序仪中于聚丙烯酰胺凝胶的单泳道中进行电泳。该方法已被证明可有效地从拟南芥（L.）Heynh的单花和单叶等小标本中检测出9种mRNA转录本，其中一些是低拷贝数的。这种方法可能很容易扩展到其他生物系统，用于发育和生理分析、群体研究及诊断。
