title: Microparticle-based RT-qPCR for highly selective rare mutation detection.
abstract: The quantitative reverse transcription polymerase chain reaction (RT-qPCR) has become one of the most widely used methods in the detection of disease-specific RNAs. The RT-qPCR involves two separate steps, RT and qPCR. In this study, we suggest a new RT-qPCR protocol with the particles of primer-immobilized networks (PINs), performing capture, RT and amplification of a target RNA in one particle. The production of undesired cDNAs was dramatically suppressed by the specific capture of the target RNA within the particle. Afterward, RT and amplification processes are performed without loss of cDNAs as exchanging the reaction solution. The biomarker gene of chronic myeloid leukemia, Bcr-Abl fusion transcript, is detected in the sensitivity of single mutant leukemic cell mixed in 104normal cell using this protocol with the excellent restraint of non-specific signal. This protocol that whole processes are performed in the particle in a row is preferred for the highly specific detection of target RNAs in complex sample.
authors: Center for BioMicrosystems, Brain Science Institute, Korea Institute of Science and Technology, Seoul 02792, Republic of Korea.
authors: Eun Hae
authors: EH
authors: Oh
authors: Center for BioMicrosystems, Brain Science Institute, Korea Institute of Science and Technology, Seoul 02792, Republic of Korea.
authors: Seungwon
authors: S
authors: Jung
authors: Department of Applied Chemistry, The Institute of Natural Science, College of Applied Science, Kyung Hee University, Yongin 17104, Republic of Korea.
authors: Won Jin
authors: WJ
authors: Kim
authors: Department of Applied Chemistry, The Institute of Natural Science, College of Applied Science, Kyung Hee University, Yongin 17104, Republic of Korea.
authors: Kwang Pyo
authors: KP
authors: Kim
authors: Center for BioMicrosystems, Brain Science Institute, Korea Institute of Science and Technology, Seoul 02792, Republic of Korea; Department of Biomedical Engineering, Korea University of Science and Technology, Daejeon 34113, Republic of Korea. Electronic address: sangk@kist.re.kr.
authors: Sang Kyung
authors: SK
authors: Kim
mesh_terms: D015374:Biosensing Techniques / Q000379:methods; D045744:Cell Line, Tumor; D017931:DNA Primers / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics; D016044:Fusion Proteins, bcr-abl / Q000235:genetics*; D006801:Humans; D061925:Immobilized Nucleic Acids / Q000235:genetics; D015464:Leukemia, Myelogenous, Chronic, BCR-ABL Positive / Q000235:genetics; D009154:Mutation; D012333:RNA, Messenger / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*
chemical_list: D017931:DNA Primers; D018076:DNA, Complementary; D061925:Immobilized Nucleic Acids; D012333:RNA, Messenger; D016044:Fusion Proteins, bcr-abl
keywords: Bcr-Abl fusion transcript; Primer-immobilized particles; RT-qPCR