应用Super SMART cDNA合成技术扩增油松胚珠总RNA

Amplifying total RNA of ovules in Pinus tabulaeformis Carr.using Super SMART cDNA synthesis technology

以油松雌性不育突变体和具有相似遗传背景可育系的胚珠为材料,用Plant RNA assistant kit试剂盒提取油松总RNA,采用Super SMART cDNA合成技术合成cDNA第l链,优化扩增第2链,通过设置梯度PCR扩增循环数,电泳分析后鉴定cDNA质量,采用定量法评估其扩增效率。可育系和不育系各1μg总RNA分别成功扩增出46.06和43.59μg纯化的双链cDNA,质量及纯度较高。此方法的应用解决了研究材料有限的问题,可由微量的胚珠总RNA扩增出足够多的高质量双链cDNA,为今后抑制性消减杂交文库的构建奠定基础。

Using ovules of the female-sterile Pinus tabulaeformis Carr.mutant and the fertile,which has the similar genetic background,as materials,the total RNA was extracted with Plant RNA assistant kit.The first-strand of cDNA synthesis and double-strands cDNA amplification were performed by Super SMART cDNA synthesis technology.The quality of cDNA was identified by gradient PCR amplification cycles and electrophoresis.Their amplification efficiency was evaluated by the quantitative method.A total of 46.06 and 43.59 μg purified double-strands cDNA were amplified successfully from 1 μg total RNA,respectively,which had high quality and purity.The results indicate that Super SMART cDNA synthesis technology could amplify enough high quality cDNA from trace of total RNA.This method solves the problem of limited research materials and builds up the foundation of suppressed subtractive hybridization in future.