几个提高抑制消减杂交(SSH)文库质量的技术改进

Improvement of Some Techniques for Enhancing Quality of Suppression Subtractive Hybridization cDNA Library

抑制消减杂交(SSH)高灵敏、易操作、高效富集低表达丰度基因和均一化目的基因片断,广泛用于差异表达基因的分离和cDNA文库构建。在实验操作中常出现低表达mRNA和cDNA丢失、大片断连接效率低等问题,构建的文库质量差,文章通过改进萃取操作提高目的产物(mRNA和cDNA)回收率,回收率增加30%以上;通过采用PVPP、高浓度KAc专一性结合和LiCl沉淀等措施除去RNA中的多酚和多糖、进而增加可分离mRNA种类和含量,对应的Uni-EST种类增加近3倍,通过采用胶回收并分段连接保护大片断cDNA等措施使文库中转录本丰富度增加,大片段(500 bp以上)比例增大,cDNA平均长度增大,文库质量明显提高。

Suppression subtractive hybridization was widely used to isolate differentially expressed genes and construct cDNA libraries at present because of its high sensitivity,easy manipulation,high efficiency in enriching rare expressed genes and normalization of targeted fragments.In the process of experiment,the regular occurrences of loss of rare expressed genes（mRNAs and their corresponding cDNAs） and low efficiency in ligation between large cDNA fragments and plasmids usually resulted in bad library.In the presented study,some improved techniques were introduced into experiments and their effects evaluated,including（i） deliberate extraction,which increased the yield of target fractionations by at least 30 %;（ii） removal of phenols and polysaccharides from RNA through specifically extraction with polyvinylpolypyrrolidone（PVPP） and high concentration of KAc as well as precipitation with LiCl, which increased concentration and species of mRNAs,and resulted in a near 3 fold increase of the corresponding Uni-ESTs;（iii） purification and fractionation cDNAs through gel electrophoresis,which increased the abundance in transcripts and the ratio of large cDNA inserters（500bp）.Due to the above measures,the quality of constructed SSH library was improved obviously.