title: Analyzing abundance of mRNA molecules with a near-infrared fluorescence technique.
abstract: This study describes a simple method for analyzing the abundance of mRNA molecules in a total DNA sample. Due to the dependence on the near-infrared fluorescence technique, this method is named near-infrared fluorescence gene expression detection (NIRF-GED). The procedure has three steps: (1) isolating total RNA from detected samples and reverse-transcription into cDNA with a biotin-labeled oligo dT; (2) hybridizing cDNA to oligonucleotide probes coupled to a 96-well microplate; and (3) detecting biotins with NIRF-labeled streptavidin. The method was evaluated by performing proof-in-concept detections of absolute and relative expressions of housekeeping and NF-κB target genes in HeLa cells. As a result, the absolute expression of three genes, Ccl20, Cxcl2, and Gapdh, in TNF-α-uninduced HeLa cells was determined with a standard curve constructed on the same microplate, and the relative expression of five genes, Ccl20, Cxcl2, Il-6, STAT5A, and Gapdh, in TNF-α-induced and -uninduced HeLa cells was measured by using NIRF-GED. The results were verified by quantitative PCR (qPCR) and DNA microarray detections. The biggest advantage of NIRF-GED over the current techniques lies in its independence of exponential or linear amplification of nucleic acids. Moreover, NIRF-GED also has several other benefits, including high sensitivity as low as several fmols, absolute quantification in the range of 9 to 147 fmols, low cDNA consumption similar to qPCR template, and the current medium throughput in 96-well microplate format and future high throughput in DNA microarray format. NIRF-GED thus provides a new tool for analyzing gene transcripts and other nucleic acid molecules.
authors: State Key Laboratory of Bioelectronics, Southeast University, Sipailou 2, Nanjing, 210096, China.
authors: Ying
authors: Y
authors: Chen
authors: Yan
authors: Y
authors: Pan
authors: Beibei
authors: B
authors: Zhang
authors: Jinke
authors: J
authors: Wang
mesh_terms: D001710:Biotin / Q000737:chemistry; D015342:DNA Probes / Q000235:genetics / Q000378:metabolism; D018076:DNA, Complementary / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression / Q000187:drug effects; D020869:Gene Expression Profiling / Q000295:instrumentation / Q000379:methods*; D020043:Genes, Essential; D006367:HeLa Cells; D006801:Humans; D016328:NF-kappa B / Q000235:genetics / Q000378:metabolism; D009838:Oligodeoxyribonucleotides / Q000235:genetics / Q000378:metabolism; D020411:Oligonucleotide Array Sequence Analysis; D012333:RNA, Messenger / Q000032:analysis* / Q000235:genetics; D048348:Reverse Transcription; D013050:Spectrometry, Fluorescence / Q000295:instrumentation / Q000379:methods*; D013055:Spectrophotometry, Infrared / Q000295:instrumentation / Q000379:methods*; D013194:Staining and Labeling; D019809:Streptavidin / Q000737:chemistry; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology
chemical_list: D015342:DNA Probes; D018076:DNA, Complementary; D016328:NF-kappa B; D009838:Oligodeoxyribonucleotides; D012333:RNA, Messenger; D014409:Tumor Necrosis Factor-alpha; C027903:oligo (dT); D001710:Biotin; D019809:Streptavidin