从非新鲜蓝氏贾第鞭毛虫中构建全长 cDNA 文库及鉴定。
Construction and characterization of a full-lengh cDNA library from non-fresh Giardia lamblia.
机构信息
School of Laboratory Medicine, Jilin Medical College, Jilin 132013, PR China.
出版信息
Asian Pac J Trop Med. 2012 Dec;5(12):931-4. doi: 10.1016/S1995-7645(12)60176-X.
OBJECTIVE
To construct rapidly a full-length cDNA library from nanogram amounts total RNA of Giardia lamblia (G. lamblia) trophozoites stocked in RNA stabilization reagent.
METHODS
Total RNA of Giardia was extracted using Trizol reagent. A full-length cDNA library of G. lamblia trophozoites was constructed by a long-distance PCR (LD-PCR) method. The recombinant rate and the coverage rate of full-length clones of the library were evaluated. The inserted fragments were identified and sequenced by PCR amplification.
RESULTS
The titer of cDNA library was 3.85 × 10(7) pfu/mL. The length of inserted fragments ranged from 0.4 to 2.5 kb, and the recombination efficiency accounted for 100% (20/20). The coverage rate of full-length clones is high (17/20).
CONCLUSIONS
The RNA stabilization reagent may be used to fix the cells and prevent the RNA in cells even though delivered under normal atmospheric temperature. The long-distance PCR can be used to construct a full-length cDNA library rapidly and it needs less RNA than the traditional method from mRNA.
目的
从贮存于 RNA 稳定剂中的蓝氏贾第鞭毛虫滋养体的纳克级总 RNA 中快速构建全长 cDNA 文库。
方法
使用 Trizol 试剂提取贾第虫总 RNA。通过长距离 PCR（LD-PCR）方法构建蓝氏贾第鞭毛虫滋养体的全长 cDNA 文库。评估文库的重组率和全长克隆的覆盖率。通过 PCR 扩增鉴定和测序插入片段。
结果
cDNA 文库的滴度为 3.85×10(7)pfu/mL。插入片段的长度范围为 0.4 至 2.5 kb，重组效率为 100%（20/20）。全长克隆的覆盖率较高（17/20）。
结论
RNA 稳定剂可用于固定细胞，防止细胞内的 RNA 在正常大气温度下降解。长距离 PCR 可用于快速构建全长 cDNA 文库，与传统方法从 mRNA 相比，所需的 RNA 量更少。
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