Molecular detection and identification of type I interferon mRNAs.
The development of a technique for identifying murine type I interferon messenger RNAs is described that involves the following essential steps: (a) the reverse transcription of total RNA extracts using oligo(dT)12-18 as a primer, (b) the amplification of any type I interferon cDNAs produced by polymerase chain reaction, and (c) the identification of interferon subtypes by hybridization of the polymerase chain reaction products to specific oligonucleotides. The technique was used to characterize the expression of the mouse interferon subtypes alpha 1, alpha 4, alpha 5, alpha 6, and beta in murine L929 cells that had been infected with Newcastle disease virus. The data derived from this study are in excellent agreement with earlier RNA protection experiments performed in the same system to characterize expression of the same genes. The present technique has advantages over those used previously, including superior sensitivity, speed, and far smaller input RNA requirements. The technique is not only applicable to other in vitro systems, but is appropriate for use in vivo.
Lai
M C
MC
Department of Microbiology, University of Western Australia, Nedlands.
Boyer
S J
SJ
Beilharz
M W
MW
D000818:Animals; D001483:Base Sequence; D007370:Interferon Type I / Q000096:biosynthesis* / Q000235:genetics; D007739:L Cells; D051379:Mice; D008969:Molecular Sequence Data; D009522:Newcastle disease virus / Q000502:physiology; D009693:Nucleic Acid Hybridization; D011089:Polydeoxyribonucleotides / Q000138:chemical synthesis; D016133:Polymerase Chain Reaction / Q000379:methods*; D012333:RNA, Messenger / Q000235:genetics / Q000302:isolation & purification*; D012194:RNA-Directed DNA Polymerase; D012680:Sensitivity and Specificity
D007370:Interferon Type I; D011089:Polydeoxyribonucleotides; D012333:RNA, Messenger; D012194:RNA-Directed DNA Polymerase