{"id":51583,"date":"2017-06-05T09:35:06","date_gmt":"2017-06-05T00:35:06","guid":{"rendered":"https:\/\/www.waseda.jp\/top\/en\/?p=51583"},"modified":"2017-06-07T09:28:37","modified_gmt":"2017-06-07T00:28:37","slug":"new-method-improving-efficiency-of-shrna-screening","status":"publish","type":"post","link":"https:\/\/www.waseda.jp\/top\/en\/news\/51583","title":{"rendered":"New method improves efficiency of shRNA screening"},"content":{"rendered":"

A group of researchers came up with a new method that improves the efficiency of short-hairpin RNA (shRNA) screening, which identifies biochemical interactions that produce pharmacological effects (known as mechanisms of action). Using this method, they found the gene that regulates cytotoxicity by aurilide B, a natural marine product that induces apoptosis, or programmed death of cells.<\/p>\n

\u201cOur new method using multiplex barcode sequencing technology facilitates the use of pooled shRNA library screening for the identification of combination drug therapy targets,\u201d says Youichi Nakao<\/a>, member of the research group and professor of biomolecular chemistry at Waseda University.<\/p>\n

\u201cCombinatorial treatment with aurilide B and oaubain, a toxic substance derived from plants, could trigger apoptosis in specific types of cancer cells with weaker effects to normal cells, suggesting a new kind of treatment.\u201d<\/p>\n

This research was published in Scientific Reports<\/em>.<\/p>\n

shRNA in cultured cells can inhibit or \u201cknown down\u201d specific genes. shRNA library is a group of various shRNA, and likewise, it creates bioactive compounds with knocked down genes in cell culture. By processing such bioactive compounds, shRNA library screening becomes possible.<\/p>\n

“Screening of genome-wide pooled shRNA libraries identifies cellular components that are functionally relevant to the mechanisms of a compound, as well as its direct targets. The Differences between control and compound-treated cell cultures in the growth rates of cells carrying a specific shRNA can be detected by deep sequencing, and this approach has a broader dynamic range than DNA micro arrays.” However, the disadvantage of this type of screening is that it is costly and has low throughput.<\/p>\n

To address this issue, the research team used multiplex barcode sequencing technology to conduct a parallel analysis of multiple samples by adding sample-specific index tags to polymerase chain reaction (PCR) primers during sequence library preparation.<\/p>\n

\u201cIn this study, we introduced a strategy for increasing the throughput of shRNA identification after screening with pooled and barcoded shRNA libraries,” explains Professor Nakao. “The addition of a sample-specific index tag to a PCR primer during sequence library preparation enabled sorting of samples analyzed in parallel by deep sequencing. We show that multiplex barcode sequencing is a reproducible, quantitative, and feasible approach for identifying molecular targets of bioactive compounds.”<\/p>\n

“We then used our method to identify genes that modify cellular sensitivity to aurilide B. The results revealed ATP1A1, which encodes a catalytic \u03b1-subunit of Na+\/K+ ATPase, as a gene involved in survival of cells treated with aurilide B. Indeed, ouabain, a known inhibitor that binds to \u03b1-subunits of Na+\/K+ ATPase, potentiated the cytotoxicity of aurilide B.”<\/p>\n

This study shows that genome-wide shRNA library screening can identify unexpected indirect target genes of bioactive compounds and that it is a novel mechanism underlying aurilide B-induced cancer cell death.<\/p>\n

Reference<\/h4>\n