title: Evaluation of the optimal provision of formalin-fixed, paraffin-embedded material for reverse transcription-PCR in soft-tissue tumour diagnosis.
abstract: OBJECTIVE
  Molecular genetic analysis is now a routine ancillary diagnostic modality to the histopathological diagnosis of soft-tissue neoplasms, many of which harbour characteristic gene fusions detectable by reverse transcription-PCR (RT-PCR). As the final diagnosis often depends on the molecular result, it is important to obtain the optimal yield of patient RNA.
  
  
  METHODS
  We assessed the most reliable method of providing formalin-fixed, paraffin-embedded material for optimal RNA yield by comparing three consecutive periods in which different preparations (5×10 μm scrolls, 5×5 μm sections and 1×10 μm sections) were used for RNA extraction for RT-PCR, with its technical success rate.
  
  
  RESULTS
  For '2011', '2012' and '2013', RT-PCR technical failure rates were 13.4%, 4.4% and 7.9%, respectively. The percentage of failed referral cases was 71.4%, 85.7% and 31.3%, and the proportion of core biopsy to excision specimens was 3:15, 2:5 and 13:3.
  
  
  CONCLUSIONS
  This study shows that the effectiveness of RNA extraction and purification is dependent on both specimen type and the tissue sectioning strategy. The failure rate has improved over recent years, particularly for large specimens as large numbers of thick 10 μm scrolls can saturate RNA extraction columns. In contrast, recent technical fails are more frequent in core biopsies, where 1×10 μm sections are insufficient for adequate RNA extraction. While previous technical fails occurred mostly in referred cases, this appears no longer the case due to the better fixation and processing of specimens in external surgical pathology departments because of the widespread recognition of the importance of molecular diagnostics as an important part of the patient pathway.
authors.affiliation: Sarcoma Unit, Royal Marsden Hospital, London, UK.
authors.forename: Khin
authors.initials: K
authors.lastname: Thway
authors.affiliation: Molecular Diagnostics, Royal Marsden Hospital, Sutton, Surrey, UK.
authors.forename: Dorte
authors.initials: D
authors.lastname: Wren
authors.affiliation: Sarcoma Unit, Royal Marsden Hospital, London, UK.
authors.forename: Jasmin
authors.initials: J
authors.lastname: Lee
authors.affiliation: Molecular Diagnostics, Royal Marsden Hospital, Sutton, Surrey, UK.
authors.forename: Lisa
authors.initials: L
authors.lastname: Thompson
authors.affiliation: Sarcoma Unit, Royal Marsden Hospital, London, UK.
authors.forename: Cyril
authors.initials: C
authors.lastname: Fisher
authors.affiliation: Molecular Diagnostics, Royal Marsden Hospital, Sutton, Surrey, UK.
authors.forename: David
authors.initials: D
authors.lastname: Gonzalez
mesh_terms: D005557:Formaldehyde; D006801:Humans; D016612:Paraffin Embedding; D057089:Pathology, Molecular; D010940:Plant Proteins; D012983:Soft Tissue Neoplasms / Q000175:diagnosis* / Q000378:metabolism / Q000473:pathology; D013048:Specimen Handling / Q000379:methods*; D016707:Tissue Fixation / Q000379:methods*
chemical_list: D010940:Plant Proteins; C077187:RT-TIP protein, Nicotiana tabacum; D005557:Formaldehyde
keywords: GENETICS; HISTOPATHOLOGY; PCR; SARCOMAS; SOFT TISSUE TUMOURS