Identification and characterization of an RNA specific primer for human tumor necrosis factor receptor I (TNFR-I).
An RNA specific primer amplified TNFR-I cDNA from cellular RNA by single-tube (ST) RT-PCR without interfering with the presence of genomic DNA of cell was identified. The primer detected TNFR-I from 1 ng of cellular RNA by ST RT-PCR, and was not cross-reacted with TNFR-II despite of the homologous sequence between TNFR-I and -II. In addition, the mechanism of the RNA specificity of the primer was investigated. An intron of 0.43 kb was inserted in the 5'-925 of TNFR-I mRNA sequence. The result corresponded with the previous determination of TNFR-I gene structure. Thus, it appears that the RNA specificity of the primer might be resulted from the antisense primer hybridizing to exon-intron junction. Owing to its high sensitivity and specificity to TNFR-I RNA, the RNA specific primer may be potentially utilized for the determination of human TNFR-I gene expression by in situ RT-PCR.
Kuo
K W
KW
Department of Biochemistry, Kaohsiung Medical College, Taiwan.
Leung
M F
MF
Leung
W C
WC
D015703:Antigens, CD / Q000235:genetics*; D004587:Electrophoresis, Agar Gel; D006801:Humans; D008970:Molecular Weight; D016133:Polymerase Chain Reaction; D012313:RNA / Q000737:chemistry* / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D018124:Receptors, Tumor Necrosis Factor / Q000235:genetics*; D047888:Receptors, Tumor Necrosis Factor, Type I; D013329:Structure-Activity Relationship; D014407:Tumor Cells, Cultured
D015703:Antigens, CD; C034477:RNA primers; D012333:RNA, Messenger; D018124:Receptors, Tumor Necrosis Factor; D047888:Receptors, Tumor Necrosis Factor, Type I; D012313:RNA