title: Real-time PCR analysis of RNA extracted from formalin-fixed and paraffin-embeded tissues: effects of the fixation on outcome reliability.
abstract: In many pathologic circumstances, quantitative mRNA expression levels are important for evaluation of possible genome mutations. The development of real-time polymerase chain reaction (RT-PCR) technology has facilitated the realization of nucleic acid quantification. Potentially, quantitative PCR offers a number of advantages over traditional methods because it permits the use of small amounts of genetic material. In the present study, we optimize a RNA purification technique on specimens that are formalin-fixed, paraffin-embedded and we examine prolonged formalin fixation effects on quantitative RT-PCR analysis. We compared RNA levels with 70 colic mucosa samples using the cyclooxygenase 2 gene as marker. The difference in amplification successes between formalin-fixed tissues and formalin-fixed, paraffin-embedded tissues was not statistically significant. Moreover, we compared the expression of formalin-fixed samples with the expression of each fresh tissue. Wilcoxon Mann-Whitney test shows that only the difference in the expression levels of 1- or 3-hour formalin-fixed samples is not statistically significant with respect to other fixation times. We found that the mRNA can be reliably extracted from formalin fixed, paraffin-embedded tissue sections but that prolonged formalin fixation produces different results in quantitative RT-PCR. It can be related to difference in RNA sequences length and the generation of secondary structures that are more susceptible to the prolonged formalin fixation. We suppose that the paraffin do not influence the RNA extraction yield because there are no statistical significant differences between amplification success of formalin-fixed tissues and paraffin-embedded tissues. Therefore, in relative expression quantization, we confirm that it is appropriate to use specimens with same protocols and time for formalin fixation.
authors.affiliation: Department of Human Pathology and Oncology, University of Florence, School of Medicine, Florence, Italy.
authors.forename: Francesca
authors.initials: F
authors.lastname: Castiglione
authors.forename: Duccio
authors.initials: D
authors.lastname: Rossi Degl'Innocenti
authors.forename: Antonio
authors.initials: A
authors.lastname: Taddei
authors.forename: Francesca
authors.initials: F
authors.lastname: Garbini
authors.forename: Anna Maria
authors.initials: AM
authors.lastname: Buccoliero
authors.forename: Maria Rosaria
authors.initials: MR
authors.lastname: Raspollini
authors.forename: Monica
authors.initials: M
authors.lastname: Pepi
authors.forename: Milena
authors.initials: M
authors.lastname: Paglierani
authors.forename: Grazia
authors.initials: G
authors.lastname: Asirelli
authors.forename: Giancarlo
authors.initials: G
authors.lastname: Freschi
authors.forename: Paolo
authors.initials: P
authors.lastname: Bechi
authors.forename: Gian Luigi
authors.initials: GL
authors.lastname: Taddei
mesh_terms: D005557:Formaldehyde / Q000737:chemistry; D006801:Humans; D009369:Neoplasms / Q000235:genetics*; D016612:Paraffin Embedding; D012334:RNA, Neoplasm / Q000032:analysis* / Q000302:isolation & purification; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000592:standards*; D013997:Time Factors; D016707:Tissue Fixation
chemical_list: D012334:RNA, Neoplasm; D005557:Formaldehyde