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Rearrangement analysis in archival thyroid tissues: punching microdissection and artificial RET/PTC 1-12 transcripts.
- Source :
-
The Journal of surgical research [J Surg Res] 2007 Dec; Vol. 143 (2), pp. 350-63. Date of Electronic Publication: 2007 Jul 26. - Publication Year :
- 2007
-
Abstract
- Background: In few papillary thyroid carcinomas (PTC) and oxyphilic thyroid carcinoma, the clinical impact of the 15 known RET hybrid oncogene variants (RET/PTC 1 to 12, 1L, 3r2, 3r3) is subject to controversial discussions. Large patient cohorts and exploitation of pathological thyroid tissue archives are essential to study the prognostic significance of RET/PTC chimeras.<br />Materials and Methods: Formalin-fixed and paraffin-embedded thyroid neoplasms were subjected to manual punching macrodissection and subsequent extraction of total RNA. Following reverse transcriptase polymerase chain reaction (RT-PCR)-based screening for RET rearrangements, hybrid-specific expression analyses were carried out for samples indicative of chimeric transcripts. Due to lack of tissue specimen harboring the rare RET chimeras, artificially constructed hybrid sequences of all known RET/PTC variants served as PCR controls.<br />Results: Manual punching dissection successfully diminished RET wild-type contamination originating from C-cells dispersed throughout normal thyroid tissues. The average amount of 27.4 mug RNA extracted allowed for repeated molecular analyses (>60 PCRs). Hybrid-specific expression analysis identified 10 of 15 RET rearrangements (8x RET/PTC 1, 2x RET/PTC 3, 5x RET/PTC x) to be found in 54 oxyphilic thyroid tumors examined. Successful amplification of each artificial hybrid sequence ensured the absence of rare chimeric transcripts. Therefore, RET/PTC x represent either common chimeras not amplifiable due to archival RNA degradation or truly novel hybrid oncoproducts.<br />Conclusions: The fast and simple techniques described here were used to examine oxyphilic carcinomas and adenomas. These microdissection and RT-PCR procedures can easily be put into practice in any molecular biology research laboratory to enable screening of large numbers of archival thyroid tumors for known as well as yet unknown RET rearrangements.
- Subjects :
- Adenoma, Oxyphilic genetics
Adenoma, Oxyphilic pathology
Adolescent
Adult
Aged
Base Sequence
Biological Specimen Banks
Carcinoma, Papillary pathology
Cell Differentiation
Chernobyl Nuclear Accident
Female
Gene Expression Regulation, Neoplastic
Humans
Male
Middle Aged
Molecular Sequence Data
Neoplasms, Radiation-Induced genetics
Neoplasms, Radiation-Induced pathology
RNA, Messenger genetics
Thyroid Neoplasms pathology
Transcription, Genetic
Carcinoma, Papillary genetics
Gene Rearrangement
Genetic Testing methods
Microdissection methods
Proto-Oncogene Proteins c-ret genetics
Thyroid Neoplasms genetics
Subjects
Details
- Language :
- English
- ISSN :
- 0022-4804
- Volume :
- 143
- Issue :
- 2
- Database :
- MEDLINE
- Journal :
- The Journal of surgical research
- Publication Type :
- Academic Journal
- Accession number :
- 17655865
- Full Text :
- https://doi.org/10.1016/j.jss.2006.10.033