A highly sensitive and reproducible HCV RNA hybridization method valuable for antiviral drug discovery.

@article{citeulike:2793603, abstract = {Real-time RT-PCR and Northern blot are employed for the measurement of HCV RNA but suffer from multiple purification steps, high cost, and relatively large variability. In this study, a hybridization method for HCV RNA detection is described. This method does not need RNA purification, and is sensitive enough to detect HCV RNA present in replicon cellular lysates harvested from a single well of a 96-well plate. Fixation of RNA by UV cross-linking is crucial for this sensitivity. A linear relationship exists between hybridization signal and cell density ranging from 10(5) to as few as 300 cells per well. The signal-to-background ratio is greater than 40 and the Z factor is above 0.7. Using several known anti-HCV agents, dose-response curves and EC(50) values generated from hybridization were similar to those obtained from a luciferase assay. This method has been successfully applied to replicons of different HCV subtypes and hepatitis B virus in our laboratory. In summary, this hybridization assay is sensitive, highly reproducible, easy to handle, and a valuable tool for antiviral drug discovery.}, address = {Achillion Pharmaceuticals, 300 George Street, New Haven, CT 06511, USA.}, author = {Zhao, Yongsen and Sanchez, Amy and Nie, Xingtie and Liu, Dongmei and Hou, Xiaohong and Fabrycki, Joanne and Phadke, Avinash and Deshpande, Milind and Huang, Mingjun and Yang, Wengang }, citeulike-article-id = {2793603}, doi = {10.1016/j.jviromet.2008.03.027}, issn = {0166-0934}, journal = {Journal of virological methods}, month = {May}, posted-at = {2008-05-13 06:35:20}, priority = {2}, title = {A highly sensitive and reproducible HCV RNA hybridization method valuable for antiviral drug discovery.}, url = {http://dx.doi.org/10.1016/j.jviromet.2008.03.027}, year = {2008} }

TY - JOUR ID - citeulike:2793603 L3 - citeulike-article-id:2793603 TI - A highly sensitive and reproducible HCV RNA hybridization method valuable for antiviral drug discovery. JF - Journal of virological methods AD - Achillion Pharmaceuticals, 300 George Street, New Haven, CT 06511, USA. SN - 0166-0934 N2 - Real-time RT-PCR and Northern blot are employed for the measurement of HCV RNA but suffer from multiple purification steps, high cost, and relatively large variability. In this study, a hybridization method for HCV RNA detection is described. This method does not need RNA purification, and is sensitive enough to detect HCV RNA present in replicon cellular lysates harvested from a single well of a 96-well plate. Fixation of RNA by UV cross-linking is crucial for this sensitivity. A linear relationship exists between hybridization signal and cell density ranging from 10(5) to as few as 300 cells per well. The signal-to-background ratio is greater than 40 and the Z factor is above 0.7. Using several known anti-HCV agents, dose-response curves and EC(50) values generated from hybridization were similar to those obtained from a luciferase assay. This method has been successfully applied to replicons of different HCV subtypes and hepatitis B virus in our laboratory. In summary, this hybridization assay is sensitive, highly reproducible, easy to handle, and a valuable tool for antiviral drug discovery. AU - Zhao, Yongsen AU - Sanchez, Amy AU - Nie, Xingtie AU - Liu, Dongmei AU - Hou, Xiaohong AU - Fabrycki, Joanne AU - Phadke, Avinash AU - Deshpande, Milind AU - Huang, Mingjun AU - Yang, Wengang PY - 2008/05/06/ UR - http://dx.doi.org/10.1016/j.jviromet.2008.03.027 ER -