Evaluation of novel formulations of 35S- and 33P-labelled nucleotides for in situ hybridization.
Radioactive in situ hybridization predominantly utilizes either RNA probes or oligonucleotide probes. The properties of various formulations of [35S]UTP alpha S have been studied with respect to probe labelling and when applied to in situ hybridization. A new formulation has been prepared that combines a high physical concentration with a high specific activity so that, theoretically, predominantly full-length, high-specific-activity primary transcripts are produced. [alpha-33P]UTP can also be used for in situ hybridization and it compares favourably to 35S-labelled probes in terms of resolution and sensitivity. Both radiolabels can also be used to label oligonucleotide probes by a tailing reaction. [35S]dATP alpha S has been reformulated specifically for labelling oligonucleotides. The traditional stabilizer, dithiothreitol, which may cause precipitation within the tailing reaction buffer, has been replaced with an alternative stabilizer that avoids this problem while maintaining the stability of the nucleotide.
Durrant
I
I
Amersham International, Buckinghamshire, UK.
Dacre
B
B
Cunningham
M
M
D000255:Adenosine Triphosphate / Q000737:chemistry / Q000378:metabolism; D000818:Animals; D005069:Evaluation Studies as Topic; D017403:In Situ Hybridization / Q000379:methods*; D007553:Isotope Labeling / Q000379:methods*; D008297:Male; D008623:Mercaptoethanol / Q000737:chemistry; D015345:Oligonucleotide Probes / Q000737:chemistry*; D010761:Phosphorus Radioisotopes / Q000737:chemistry* / Q000378:metabolism; D010902:Pituitary Gland / Q000166:cytology; D051381:Rats; D017208:Rats, Wistar; D012680:Sensitivity and Specificity; D013462:Sulfur Radioisotopes / Q000737:chemistry* / Q000378:metabolism; D014544:Uridine Triphosphate / Q000737:chemistry / Q000378:metabolism
D015345:Oligonucleotide Probes; D010761:Phosphorus Radioisotopes; D013462:Sulfur Radioisotopes; D008623:Mercaptoethanol; D000255:Adenosine Triphosphate; D014544:Uridine Triphosphate