Simultaneous determination of multiple mRNA levels utilizing MALDI-TOF mass spectrometry and biotinylated dideoxynucleotides.
Here we report an efficient method to simultaneously measure multiple mRNA levels utilizing mass spectrometry (MS) and molecular affinity isolation. In this approach, reverse transcription products of a group of mRNAs are subjected to competitive PCR with competitors and internal standards of known concentrations, and the PCR products are differentiated and quantified by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS to determine the mRNA levels. The method provides high accuracy in quantitative MS analysis due to the facilitated purification of oligonucleotides by molecular affinity isolation. Additionally, owing to the molecular affinity isolation, only those oligonucleotides required for expression level determination are introduced into the mass spectrometer, while other irrelevant reaction components that could overlap with peaks of gene transcripts or competitors are removed prior to MS analysis. Thus the approach enhances the parallel analysis of multiple gene transcripts by MS. Utilizing the method we have simultaneously measured mRNA levels of four genes (Rho, Nrl, Hprt, and Lhx2) in mouse retinal tissue.
Duffield
Daniel Scott
DS
Department of Chemical and Biochemical Engineering, Rutgers University, Piscataway, New Jersey 08854, USA.
Cai
Li
L
Kim
Sobin
S
D000818:Animals; D001483:Base Sequence; D050976:Basic-Leucine Zipper Transcription Factors / Q000235:genetics; D019921:Biotinylation; D054306:Dideoxynucleotides; D005136:Eye Proteins / Q000235:genetics; D018398:Homeodomain Proteins / Q000235:genetics; D007041:Hypoxanthine Phosphoribosyltransferase / Q000235:genetics; D060850:LIM-Homeodomain Proteins; D051379:Mice; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods; D012313:RNA; D012333:RNA, Messenger / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D012194:RNA-Directed DNA Polymerase; D012160:Retina / Q000502:physiology; D012234:Rho Factor / Q000235:genetics; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D014157:Transcription Factors / Q000235:genetics; D014158:Transcription, Genetic
D050976:Basic-Leucine Zipper Transcription Factors; D054306:Dideoxynucleotides; D005136:Eye Proteins; D018398:Homeodomain Proteins; D060850:LIM-Homeodomain Proteins; C489784:Lhx2 protein, mouse; C084884:Nrl protein, mouse; C034477:RNA primers; D012333:RNA, Messenger; D012234:Rho Factor; D014157:Transcription Factors; D012313:RNA; D007041:Hypoxanthine Phosphoribosyltransferase; D012194:RNA-Directed DNA Polymerase
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