Improved design of riboprobes from pBluescript and related vectors for in situ hybridization.
The pBluescript family of plasmids and phagemids are sophisticated multi-purpose cloning vectors that allow convenient production of single-stranded sense and anti-sense RNA probes corresponding to DNA sequences inserted into a large multiple cloning site array. We have observed that in many applications sense (control) probes generated from genes cloned into pBluescript II KS(-) give high background signals on in situ hybridization to human tissue sections. Our studies indicate that this spurious hybridization is due to sequences contained within both strands of the multiple cloning site between the SmaI and SacI sites that are similar to human 28S rRNA. This information is useful in construct design in order to minimize nonspecific background problems, as demonstrated by in situ hybridization of sense and anti-sense probes corresponding to a portion of human stromelysin-3 to sections of human lung carcinoma.
Witkiewicz
H
H
Department of Orthopedics, Mayo Clinic, Rochester, MN 55905.
Bolander
M E
ME
Edwards
D R
DR
D001483:Base Sequence; D005822:Genetic Vectors; D006801:Humans; D017403:In Situ Hybridization / Q000379:methods*; D008175:Lung Neoplasms / Q000235:genetics; D053507:Matrix Metalloproteinase 11; D008666:Metalloendopeptidases / Q000235:genetics; D015336:Molecular Probe Techniques; D008969:Molecular Sequence Data; D009363:Neoplasm Proteins / Q000235:genetics; D014066:Palatine Tonsil / Q000378:metabolism; D010957:Plasmids; D015347:RNA Probes; D012339:RNA, Ribosomal, 28S / Q000235:genetics
D009363:Neoplasm Proteins; D015347:RNA Probes; D012339:RNA, Ribosomal, 28S; D053507:Matrix Metalloproteinase 11; D008666:Metalloendopeptidases