ABSTRACT
Objective To establish RNase L gene knockout HEK 293 cell lines using CRISPR/Cas9 system.Methods Small guide RNA ( sgRNA) sequences of human RNase L were designed and sgRNAs were inserted into pCas-Guide and pCas-guide RNA(gRNA) vectors were obtained.The donor DNA sequences of the homologous arm were designed for RNase L knockout .In the presence of the right homologous arm , the resistance gene of hygromycin B and the left homologous arm as templates of homology-directed repair , the donor DNA template was amplified by overlopping PCR and cloned into the pBackZero-T expression vector and pBackZero-T-RNase LK vector was obtained .The pCas-gRNA vector and pBackZero-T-RNase LK vector were co-transfected into HEK293 cells to establish the stable expression cell line of RNase L gene knockout .Cells were cultured with hygromycin B , while Western blotting and DNA sequencing were used to analyze the gene of RNase L knockout from genome .Results and Conclusion The pCas-gRNA vector and pBackZero-T-RNase LK vector were successfully constructed.Five RNase L gene knockout HEK293 cell lines were generated,contributing to the study of the biological function and molecular mechanism of RNase L .
ABSTRACT
Objective To develop and optimize a new method to extract miRNAs from plasma.Methods miRNAs were extracted from plasma by mixing it with the extraction solution that contained surfactant and by heating .Then the ribonuclease inhibitor was added into the extraction to prevent RNAs from degradation .The expression level of each miRNA was detected by real-time quantitative PCR in oder to evaluate the feasibility of this method .Results A method which extracted miRNAs from plamsa in just one step was established .The specificity , reproducibility and stability of this method have been demonstrated by real-time quantitative PCR .Conclusion The one-step method is simple , inexpensive , and plasma-saving.It seems like a new method for clinical examination of miRNAs from plasma .