Detection of Fusion Gene Transcripts in Fresh-Frozen and Fromalin-Fixed Paraffin-Embedded Tissue Sections of Soft-Tissue Sarcomas after Laser Capture Microdissection and RT-PCR

Author(s): Jin L, Majerus A, Oliveira C, Inwards Y, Nascimento G, Burgart J, Lloyd R V

Publication: Diagn Mol Pathol, 2003, Vol. 12, Page 224

PubMed ID: 14639108 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   To evaluate the sensitivity of fresh frozen and paraffin embedded (PE) tisue sections for RT-PCR based chimera gene detection after cell microdissection of sarcoma specimens.

   Summary of Findings:

   The sensitivity of RT-PCR detection of the fusion gene transcript was 10 to 50-fold greater in fresh frozen compared to PE tissue. Fusion transcripts were detected using as few as 50 or 200 microdissected sarcoma cells in frozen and PE tissue sections, respectively. Subsequent Southern blot analysis increased sensitivity in comparison to ethidium bromide gel staining.

   Biospecimens

   • Tissue - Lung

   Preservative Types

   • Frozen

   Diagnoses:

   • Neoplastic - Sarcoma

   Platform:

   Analyte	Technology Platform
   RNA	RT-PCR
   DNA	Southern blot

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Biospecimen Aliquots and Components	Cell number	1 cell 10 cells 50 cells 200 cells 1000 cells
   Biospecimen Preservation	Embedding medium	Paraffin Fresh frozen

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2. Study Purpose

   To determine the PCR dectection threshold (ie. cycle number) for fusion transcripts in cells microdissected from frozen and PE tissue sections.

   Summary of Findings:

   Fusion transcripts were detected after 40 PCR cycles and Southern blot analysis using as few as 50 and 200 cells isolated from fresh frozen and PE tissue, respectively. However, weaker bands were observed after 35 PCR cycles when a larger number of cells were analyzed, 200 and 1000 cells from fresh frozen and PE tissue, respectively.

   Biospecimens

   • Tissue - Lung

   Preservative Types

   • Frozen

   Diagnoses:

   • Neoplastic - Sarcoma

   Platform:

   Analyte	Technology Platform
   DNA	Southern blot
   RNA	RT-PCR

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Biospecimen Preservation	Embedding medium	Paraffin Fresh frozen
   Biospecimen Aliquots and Components	Cell number	1 cell 10 cells 50 cells 200 cells 1000 cells
   RT-PCR Specific	Number of cycles	30 cycles 35 cycles 40 cycles

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