title: Effect of formalin, acetone, and RNAlater fixatives on tissue preservation and different size amplicons by real-time PCR from paraffin-embedded tissue.
abstract: RNA recovered from paraffin-embedded tissue has been reported to be a suitable substrate for polymerase chain reaction. During tissue fixation and paraffin embedding, RNA undergoes degradation, but with certain restrictions, it can be used for gene expression studies. At the same time, formalin-fixed, paraffin embedded histopathology archives contain an unestimable collection, which could be analyzed to investigate changes in mRNA expression in pathologic processes. To decide for future tissue conservation of pathology samples, it would be reasonable to satisfy both histologic and molecular biologic needs. The effect of three different fixation methods, RNAlater (SIGMA R 0901, St Louis, MO), acetone, and formalin, were compared by histology, immunohistochemistry, and real-time PCR. To assess tissue structure preservation and antigenicity, hematoxylin-eosin staining and immunohistochemistry were performed; to assess RNA quality, RNA was extracted and the transcription of different amplicon sizes (121, 225, 406 bp for GAPDH; 166, 310, 536 bp for beta globin) were examined on human endometrium samples. The most adequate tissue preservation was found in case of formalin fixation, while there were no significant differences in the three fixatives' yields for various size real-time PCR amplicons. Longer amplicons (above approximately 225 bp) have limited use for gene expression studies, while shorter amplicons could give more reliable results.
authors.affiliation: 2nd Department of Pathology, Semmelweis University, Budapest, Hungary. paskacsill@excite.com
authors.forename: Csilla
authors.initials: C
authors.lastname: Páska
authors.forename: Krisztina
authors.initials: K
authors.lastname: Bögi
authors.forename: László
authors.initials: L
authors.lastname: Szilák
authors.forename: Annamária
authors.initials: A
authors.lastname: Tokés
authors.forename: Erzsébet
authors.initials: E
authors.lastname: Szabó
authors.forename: István
authors.initials: I
authors.lastname: Sziller
authors.forename: János
authors.initials: J
authors.lastname: Rigó
authors.forename: Gábor
authors.initials: G
authors.lastname: Sobel
authors.forename: István
authors.initials: I
authors.lastname: Szabó
authors.forename: Pál
authors.initials: P
authors.lastname: Kaposi-Novák
authors.forename: András
authors.initials: A
authors.lastname: Kiss
authors.forename: Zsuzsa
authors.initials: Z
authors.lastname: Schaff
mesh_terms: D000328:Adult; D014408:Biomarkers, Tumor / Q000378:metabolism; D004717:Endometrium / Q000378:metabolism / Q000473:pathology / Q000601:surgery; D005260:Female; D005784:Gene Amplification; D006801:Humans; D007124:Immunoenzyme Techniques; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D019188:Organ Preservation Solutions; D016612:Paraffin Embedding; D012313:RNA / Q000302:isolation & purification*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D014591:Uterine Diseases / Q000378:metabolism / Q000473:pathology / Q000601:surgery
chemical_list: D014408:Biomarkers, Tumor; D019188:Organ Preservation Solutions; D012313:RNA