In situ hybridization using cRNA probes: isotopic and nonisotopic detection methods.
In this chapter we describe the use of cRNA (riboprobes) in the detection of gene expression in tissue sections. Riboprobes offer good sensitivity and allow the detection of low-level mRNA expression. In some cases, the use of radiolabeling is justified because this method is still sensitive. However, recent advances in nonisotopic detection methods mean that in some cases digoxigenin (DIG) or biotin labeling also may be sufficiently sensitive to detect mRNA expression in tissues of interest. The use of alkaline phosphatase conjugated anti-DIG antibodies improves the sensitivity of DIG detection over peroxidase systems, and the use of amplification systems based on biotinyl tyramide has improved the sensitivity of biotin labelled probe detection. Finally, it can be shown that low-level mRNA expression is easier to detect in frozen sections than in paraffin-embedded material, with a consequent loss in quality of morphology.
Darby
Ian A
IA
School of Medical Sciences, RMIT University, Melbourne, Australia.
Bisucci
Teresa
T
Desmoulière
Alexis
A
Hewitson
Tim D
TD
D000818:Animals; D001345:Autoradiography; D001710:Biotin / Q000031:analogs & derivatives / Q000378:metabolism; D004076:Digoxigenin / Q000378:metabolism; D006801:Humans; D017403:In Situ Hybridization / Q000379:methods*; D021141:Nucleic Acid Amplification Techniques; D009693:Nucleic Acid Hybridization / Q000379:methods*; D018075:RNA, Complementary / Q000235:genetics* / Q000378:metabolism*; D012333:RNA, Messenger / Q000032:analysis*; D012680:Sensitivity and Specificity; D014439:Tyramine / Q000031:analogs & derivatives / Q000378:metabolism
D018075:RNA, Complementary; D012333:RNA, Messenger; C111551:biotinyltyramide; D001710:Biotin; D004076:Digoxigenin; D014439:Tyramine