Non-radioactive in situ hybridization for mRNA with emphasis on the use of oligodeoxynucleotide probes

Abstract

Synthetic oligodeoxynucleotides represent attractive tools for mRNA in situ hybridization. In the present review, guidelines for the preparation, labeling and use of such probes are discussed. Emphasis is placed on sensitivity and specificity issues.

Downregulation of Bcl-2 by Podocytes Is Associated with Progressive Glomerular Injury and Clinical Indices of Poor Renal Prognosis in Human IgA Nephropathy

“…The use of DIGlabeled oligonucleotide probe cocktails in nonradioactive in situ hybridization method have been reported to have a detection sensitivity akin to methods that use radioisotope-labeled antisense RNA probes (24,25). This detection sensitivity can be boosted by incorporating the signal amplification systems (26,27). Every probe cocktail consisted of an equimolar mixture of three to four individual single-stranded DNA antisense oligonucleotides, each 27 to 31 bp long, with DIG labeling at both ends.…”

Simultaneous Quantification of Active Carbon- and Nitrogen-Fixing Communities and Estimation of Fixation Rates Using Fluorescence In Situ Hybridization and Flow Cytometry

“…It should be noted that the equation used to determine the specificity of oligonucleotide probes, at its most stringent, allows for 20% mismatch (47). Cell suspensions were incubated at 35°C for 1 h in hybridization buffer containing 1 to 150 l formamide, 30 l 20ϫ sodium citrate buffer (SSC; 1ϫ SSC is 0.15 M NaCl plus 0.015 M sodium citrate; Amresco), 60 l 10%, wt/vol, dextran sulfate, 30 l 10%, wt/vol, blocking solution (component D; TSA kit 6, T-20916; Invitrogen), 15 l 4 mg ml Ϫ1 yeast RNA, 6 l 10 mg ml…”

Detection of mRNA of Kinesin Superfamily 3A in the Cerebrum and Cerebellum. Biotin-Tyramine-Catalyzed Signal Amplification for In Situ Hybridization.

“…Because they could be readily obtained from a com- mercial source, we chose to conduct this study using oligonucleotide probes: earlier studies had shown that ISH could be successfully carried out using such oligonucleotide probes [6,11,14]. The sequence data of selected genes were obtained from the GenBank internet site, and the sequences of the probes are designed using commercially available computer software.…”

“…Because there is a 1:1 stoichiometry between the label and the probe, the sensitivity was relatively low compared to some other labeling protocols. Therefore, to increase the sensitivity, we used a cocktail of two probes that were bound to separate sites at the head and tail regions of the targeted mRNA [6].…”

“…The probes were designed using GENETYX ver. 6.03 software running on a Macintosh computer as previously described [6], making certain that our probes did not hybridize with sequences from any other members of the kinesin superfamily in the database. Two sense and two antisense probes were constructed: one for the head region of the KIF3A cDNA sequence (sense, 5'-biotin-GGT-…”