In Situ Hybridization Using the bDNA Technology
The branched DNA (bDNA) technology has been used quite extensively in the microwell format to determine the levels of viral nucleic acids, as well as specific cellular RNAs [1, 2]. The bDNA technology, which is based on signal amplification, is easy to use, and does not require the use of radioactivity. With the microwell format, it has been shown to be sensitive and specific. The technology offers the additional benefits of a wide dynamic range, as well as accurate and precise quantification. We have recently applied the bDNA technology to the in situ detection of nucleic acids. In situ hybridization allows for the identification of individual cells that have a higher (or lower) level of a particular nucleic acid sequence, in the background of many cells that have normal levels. In addition, in situ hybridization also gives the morphological context of these “positive” cells, which in many applications is very important.
KeywordsTarget Probe Moist Chamber Viral Nucleic Acid Normal Human Bronchial Epithelial Nucleic Acid Target
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- 1.Kolberg JA, Ludtke DN, Shen L-P, Cao W, O’Conner D, Urdea MS, Wuestehube LJ, Lewis M. Branched DNA (bDNA) technology for direct quantification of nucleic acids: design and performance. In: Ferre F, ed. Gene Quantification. Boston: Birkhauser, 1998.Google Scholar
- 3.Collins M, Irvine B, Tyner D, Fine E, Zayati C, Chang C, Horn T, Ahle D, Detmer J, Shen L-P, Kolberg J, Bushnell S, Urdea M, Ho D. A branched DNA signal amplification assay for quantification of nucleic acid targets below 100 molecules/ml. Nucl Acids Res 1997; 25: 2979–2984.PubMedCrossRefGoogle Scholar
- 4.Barr S, Fusaro R, Gorrin G, Hendricks D, Kolberg J, Robertson J, Shen L, Irvine B. Presented at the 12th World AIDS Conference, Geneva, Switzerland, June 28–July 3, 1998. Clin Sci 1998; 2: 647–651.Google Scholar
- 5.Cao W, Collins M, Ng S, Chang C, Thompson P, Kolberg J. Poster presented at the 12th Annual San Diego Conference, The American Association for Clinical Chemistry, November 6–8, 1997, San Diego, CA.Google Scholar
- 8.Cao W, Connolly J, Zagala M, Beard C, Hirsch A, Ku L, Kolberg J, Teramoto Y. Proceedings of the American Association for Cancer Research Eighty-Ninth Annual Meeting, New Orleans, LA. 1998; 39: 335.Google Scholar
- 9.Zhou J, Mulshine JL, Unsworth EJ, Scott FM, Avis IM, Vos MD, Treston AM. Purification and characterization of a protein that permits early detection of lung cancer. Identification of heterogeneous nuclear ribonucleoprotein A2/B1 as the antigen for monclonal antibody 703D4. J Biol Chem 1996; 271: 10760–10766.PubMedCrossRefGoogle Scholar
- 10.Tockman MS, Mulshine JL, Piantadosi S, Erozan YS, Gupta PK, Ruckdeschel JC, Taylor PR, Zhukov T, Zhou W-H, Qiao Y-L, Yao S-X for the Lung Cancer Early Detection Work Group Investigators and the Yunnan Tin Corporation Investigators. Prospective detection of preclinical lung cancer: results from two studies of heterogeneous nuclear ribonucleoprotein A2B 1 overexpression. Clin Cancer Res 1997; 3: 2237–2246.PubMedGoogle Scholar
- 11.Grogan T, et al. 1999;(manuscript in preparation).Google Scholar