A simple method based on PCR for detecting the relative mRNA amounts of the four mouse IgG subclasses.
A simple method based on the polymerase chain reaction (PCR) was developed for detecting the relative mRNA amounts of the four mouse IgG subclasses in total RNA samples and is described in this report. The main features of this method are, first, cDNA amplification including VH through the constant region(CH2 domain) of each IgG subclass with a set of consensus PCR primers(VH1BACK and 32P-labeled C gamma 32), and secondly, cleavage of the amplified DNA fragments with BamHI and XhoI endonucleases which act at distinct cleavage sites in the constant region of each IgG subclass. The radioactive intensities of the different sized fragments separated on polyacrylamide gel were used to show the relative amounts of IgG subclasses at the RNA level. This method provides clear detection of each IgG subclass using RNA samples from tissues in which Ig-producing cells are rare.
Ono
M
M
Department of Pathology, Tohoku University School of Medicine, Sendai, Japan.
Yamamoto
T
T
Nose
M
M
D000818:Animals; D001483:Base Sequence; D017931:DNA Primers; D007074:Immunoglobulin G / Q000032:analysis* / Q000145:classification / Q000235:genetics; D007421:Intestine, Small / Q000276:immunology; D051379:Mice; D008810:Mice, Inbred C57BL; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*; D012333:RNA, Messenger / Q000032:analysis*; D013154:Spleen / Q000276:immunology
D017931:DNA Primers; D007074:Immunoglobulin G; D012333:RNA, Messenger