Hybridisation of [76Br]-labelled antisense oligonucleotides to Chromogranin A mRNA verified by RT-PCR.
Methods have been developed to label oligonucleotides (ODNs) in the 5'-position with (76)Br via a prosthetic group on a hexylamino-linker. The purpose of the study was to explore whether the labelling procedure would prevent specific hybridisation by using reverse transcription-polymerase chain reaction (RT-PCR) followed by sequencing of the PCR product. Antisense ODNs (30 mer, specific for rat Chromogranin A [CgA] mRNA) with phosphodiester (O-ODN) or phosphothioate (S-ODN) backbone, either unlabelled or labelled with (76)Br, served as one of the primers in individual PCR reactions. Using O-ODN as a primer, irrespective of being labelled or not, a selected 225-bp PCR fragment was successfully amplified. However, no amplification was obtained using S-ODN as a primer. The proper PCR products were only detected in the sample prepared from the adrenal gland, but not in that from the heart, liver or kidney. Autoradiographic recording of the gel, after gel electrophoresis, revealed radioactive signals corresponding to the amplified PCR products. The sequence of the PCR product matched the rat CgA mRNA sequence obtained from the EMBL database. RT-PCR is an attractive method to identify the selective binding of modified ODNs to target mRNA. This method confirmed that the labelling with (76)Br did not change the hybridisation ability of antisense O-ODN.
Wu
Feng
F
Uppsala IMANET AB, PO Box 967, 751 85 Uppsala, Sweden.
Lendvai
Gabor
G
Yngve
Ulrika
U
Eriksson
Barbro
B
Långström
Bengt
B
Bergström
Mats
M
D000818:Animals; D001967:Bromine Radioisotopes / Q000493:pharmacokinetics* / Q000627:therapeutic use; D053379:Chromogranin A; D002864:Chromogranins / Q000235:genetics*; D020868:Gene Silencing; D007553:Isotope Labeling / Q000379:methods; D008297:Male; D009693:Nucleic Acid Hybridization / Q000235:genetics / Q000379:methods; D009841:Oligonucleotides / Q000493:pharmacokinetics* / Q000627:therapeutic use; D016376:Oligonucleotides, Antisense / Q000235:genetics* / Q000493:pharmacokinetics* / Q000627:therapeutic use; D009928:Organ Specificity; D012333:RNA, Messenger / Q000235:genetics*; D019275:Radiopharmaceuticals / Q000493:pharmacokinetics* / Q000627:therapeutic use; D051381:Rats; D017207:Rats, Sprague-Dawley; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D014018:Tissue Distribution
D001967:Bromine Radioisotopes; D053379:Chromogranin A; D002864:Chromogranins; D009841:Oligonucleotides; D016376:Oligonucleotides, Antisense; D012333:RNA, Messenger; D019275:Radiopharmaceuticals; C506153:chromogranin A, rat