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.
Miyazaki
M
M
Department of Nephrology, Tokai University School of Medicine, Isehara.
Nikolic-Paterson
D J
DJ
Endoh
M
M
Nomoto
Y
Y
Sakai
H
H
Atkins
R C
RC
Koji
T
T
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