A sensitive method of non-radioactive in situ hybridization for mRNA localization within human renal biopsy specimens: use of digoxigenin labeled oligonucleotides.
A sensitive method of non-radioactive in situ hybridization using digoxigenin-labeled oligonucleotides is described for the detection of mRNA within human renal biopsy specimens. Although non-radioactive in situ hybridization typically has the drawback of low sensitivity, we increased the sensitivity of this method, providing a practical alternative to the use of radiolabelled probes. The four main points are: 1) assessment of the efficiency of labeling, 2) optimization of the probe concentration for hybridization, 3) requirement of deproteinization of tissues with HCl and proteinase K, and 4) the use of a four-layer immunoperoxidase staining system. This technique was found to clearly localize individual mRNA positive cells within cryostat tissue sections. A variety of controls including sense probes, excess unlabeled anti-sense probes, and RNase-treatment demonstrated the specificity of the technique. This improved method is a powerful technique for detecting mRNA within human renal tissue and will be most useful in the study of gene expression in the pathogenesis of renal diseases.
Department of Nephrology, Tokai University School of Medicine, Isehara.
M
M
Miyazaki
D J
DJ
Nikolic-Paterson
M
M
Endoh
Y
Y
Nomoto
H
H
Sakai
R C
RC
Atkins
T
T
Koji
D000199:Actins / Q000096:biosynthesis / Q000235:genetics; D001483:Base Sequence; D001706:Biopsy; D004076:Digoxigenin; D005922:Glomerulonephritis, IGA / Q000235:genetics / Q000473:pathology; D006801:Humans; D007124:Immunoenzyme Techniques; D017403:In Situ Hybridization / Q000379:methods*; D015850:Interleukin-6 / Q000096:biosynthesis / Q000235:genetics; D007668:Kidney / Q000737:chemistry* / Q000473:pathology; D008969:Molecular Sequence Data; D015345:Oligonucleotide Probes; D012333:RNA, Messenger / Q000032:analysis*; D012680:Sensitivity and Specificity
D000199:Actins; D015850:Interleukin-6; D015345:Oligonucleotide Probes; D012333:RNA, Messenger; D004076:Digoxigenin