title: Quantitative assessment of short amplicons in FFPE-derived long-chain RNA.
abstract: Formalin-fixed paraffin-embedded (FFPE) tissues are important resources for molecular medical research. However, long-chain RNA analysis is restricted in FFPE tissues due to high levels of degradation. To explore the possibility of long RNA quantification in FFPE tissues, we selected 14 target RNAs (8 mRNAs and 6 long noncoding RNAs) from literatures, and designed short (~60 bp) and long (~200 bp) amplicons for each of them. Colorectal carcinomas with adjacent normal tissues were subjected to quantitative reverse-transcription PCR (quantitative RT-PCR) in 3 cohorts, including 18 snap-frozen and 83 FFPE tissues. We found that short amplicons were amplified more efficiently than long amplicons both in snap-frozen (P = 0.0006) and FFPE (P = 0.0152) tissues. Nonetheless, comparison of colorectal carcinomas with their adjacent normal tissues demonstrated that the consistency of fold-change trends in a single short amplicon between snap-frozen and FFPE tissues was only 36%. Therefore, we innovatively performed quantitative RT-PCR with 3 non-overlapping short amplicons for 14 target RNAs in FFPE tissues. All target RNAs showed a concordance of 100% of fold-change trends in at least two short amplicons, which offers sufficient information for accurate quantification of target RNAs. Our findings demonstrated the possibility of long-chain RNA analysis with 3 non-overlapping short amplicons in standardized-preserved FFPE tissues.
authors.affiliation: Department of Pathology, Shanghai Medical College, Fudan University, Shanghai, 200032, China.
authors.forename: Hui
authors.initials: H
authors.lastname: Kong
authors.affiliation: Harvard College, Harvard University, Cambridge, MA, 02138, USA.
authors.forename: Mengou
authors.initials: M
authors.lastname: Zhu
authors.affiliation: Department of Pathology, Shanghai Medical College, Fudan University, Shanghai, 200032, China.
authors.forename: Fengyun
authors.initials: F
authors.lastname: Cui
authors.affiliation: Department of Pathology, Shanghai Medical College, Fudan University, Shanghai, 200032, China.
authors.forename: Shuyang
authors.initials: S
authors.lastname: Wang
authors.affiliation: Department of Pathology, Huashan Hospital, Fudan University, Shanghai, 200040, China.
authors.forename: Xue
authors.initials: X
authors.lastname: Gao
authors.affiliation: Department of Pathology, Zhongshan Hospital, Fudan University, Shanghai, 200032, China.
authors.forename: Shaohua
authors.initials: S
authors.lastname: Lu
authors.affiliation: Department of Pathology, Shanghai Medical College, Fudan University, Shanghai, 200032, China.
authors.forename: Ying
authors.initials: Y
authors.lastname: Wu
authors.affiliation: 1] Department of Pathology, Shanghai Medical College, Fudan University, Shanghai, 200032, China [2] Department of Pathology, Huashan Hospital, Fudan University, Shanghai, 200040, China [3] Institute of Biomedical Sciences, Fudan University, Shanghai, 200032, China.
authors.forename: Hongguang
authors.initials: H
authors.lastname: Zhu
mesh_terms: D003110:Colonic Neoplasms / Q000235:genetics; D015179:Colorectal Neoplasms / Q000235:genetics; D005557:Formaldehyde / Q000737:chemistry; D006801:Humans; D016612:Paraffin Embedding / Q000379:methods; D012313:RNA / Q000235:genetics*; D012004:Rectal Neoplasms / Q000235:genetics
chemical_list: D005557:Formaldehyde; D012313:RNA