Scalable transcriptional analysis routine--multiplexed quantitative real-time polymerase chain reaction platform for gene expression analysis and molecular diagnostics.
We report the development of a new technology for simultaneous quantitative detection of multiple targets in a single sample. Scalable transcriptional analysis routine (STAR) represents a novel integration of reverse transcriptase-polymerase chain reaction and capillary electrophoresis that allows detection of dozens of gene transcripts in a multiplexed format using amplicon size as an identifier for each target. STAR demonstrated similar or better sensitivity and precision compared to two commonly used methods, SYBR Green-based and TaqMan probe-based real-time reverse transcriptase-polymerase chain reaction. STAR can be used as a flexible platform for building a variety of applications to monitor gene expression, from single gene assays to assays analyzing the expression level of multiple genes. Using severe acute respiratory syndrome (SARS) corona virus as a model system, STAR technology detected single copies of the viral genome in a two-gene multiplex. Blinded studies using RNA extracted from various tissues of a SARS-infected individual showed that STAR correctly identified all samples containing SARS virus and yielded negative results for non-SARS control samples. Using alternate priming strategies, STAR technology can be adapted to transcriptional profiling studies without requiring a priori sequence information. Thus, STAR technology offers a flexible platform for development of highly multiplexed assays in gene expression analysis and molecular diagnostics.
Garcia
Elizabeth P
EP
Primera BioSystems, Providence, RI 02906, USA.
Dowding
Lori A
LA
Stanton
Lawrence W
LW
Slepnev
Vladimir I
VI
D000818:Animals; D001921:Brain / Q000378:metabolism; D003116:Color; D005456:Fluorescent Dyes; D020869:Gene Expression Profiling / Q000295:instrumentation / Q000379:methods*; D012367:RNA, Viral / Q000032:analysis / Q000235:genetics; D051381:Rats; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000295:instrumentation / Q000379:methods*; D045473:Severe acute respiratory syndrome-related coronavirus / Q000235:genetics / Q000302:isolation & purification; D012680:Sensitivity and Specificity; D045169:Severe Acute Respiratory Syndrome / Q000175:diagnosis* / Q000821:virology; D019914:Taq Polymerase / Q000378:metabolism; D013997:Time Factors; D014158:Transcription, Genetic / Q000235:genetics*
D005456:Fluorescent Dyes; D012367:RNA, Viral; D019914:Taq Polymerase