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

ObjectiveTo construct rapidly a full–length cDNA library from nanogram amounts total RNA of Giardia lamblia (G. lamblia) trophozoites stocked in RNA stabilization reagent.MethodsTotal 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.ResultsThe titer of cDNA library was 3.85 × 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).ConclusionsThe 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.