Construction and characterization of a full-lengh cDNA library from non-fresh Giardia lamblia

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• 作者：Jun-Li Guo^a ; ^b ; ^¦¤ ; Jie Jiang^a ; ^b ; ^¦¤ ; Wen-Yu Zheng^c ; ^¦¤ ; Ming-Luan Li^b ; ^¦¤ ; Xi-Feng Tian^d ; Xian-Min Feng^a ; ^{xianminfeng@yahoo.cn} ; Yue-Hua Wang^a ; Xiao-Hong Ju^a ; Yue-Qiong Kong^b
• 关键词：Giardia lamblia ; cDNA library ; RNA stabilization reagent
• 刊名：Asian Pacific Journal of Tropical Medicine
• 出版年：2012
• 出版时间：December, 2012
• 年：2012
• 卷：5
• 期：12
• 页码：931-934
• 全文大小：580 K

文摘

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 ¡Á 10⁷ 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.