title: The search for an optimal DNA, RNA, and protein detection by in situ hybridization, immunohistochemistry, and solution-based methods.
abstract: Clinical trials and correlative laboratory research are increasingly reliant upon archived paraffin-embedded samples. Therefore, the proper processing of biological samples is an important step to sample preservation and for downstream analyses like the detection of a wide variety of targets including micro RNA, DNA and proteins. This paper analyzed the question whether routine fixation of cells and tissues in 10% buffered formalin is optimal for in situ and solution phase analyses by comparing this fixative to a variety of cross linking and alcohol (denaturing) fixatives. We examined the ability of nine commonly used fixative regimens to preserve cell morphology and DNA/RNA/protein quality for these applications. Epstein-Barr virus (EBV) and bovine papillomavirus (BPV)-infected tissues and cells were used as our model systems. Our evaluation showed that the optimal fixative in cell preparations for molecular hybridization techniques was "gentle" fixative with a cross-linker such as paraformaldehyde or a short incubation in 10% buffered formalin. The optimal fixatives for tissue were either paraformaldehyde or low concentration of formalin (5% of formalin). Methanol was the best of the non cross-linking fixatives for in situ hybridization and immunohistochemistry. For PCR-based detection of DNA or RNA, some denaturing fixatives like acetone and methanol as well as "gentle" cross-linking fixatives like paraformaldehyde out-performed other fixatives. Long term fixation was not proposed for DNA/RNA-based assays. The typical long-term fixation of cells and tissues in 10% buffered formalin is not optimal for combined analyses by in situ hybridization, immunohistochemistry, or--if one does not have unfixed tissues--solution phase PCR. Rather, we recommend short term less intense cross linking fixation if one wishes to use the same cells/tissue for in situ hybridization, immunohistochemistry, and solution phase PCR.
authors.affiliation: Divisions of Hematology & Medical Oncology, Department of Internal Medicine, Comprehensive Cancer Center, College of Medicine, The Ohio State University, 320 West 10th Avenue, Columbus, OH 43210, USA.
authors.forename: Fengting
authors.initials: F
authors.lastname: Yan
authors.forename: Xin
authors.initials: X
authors.lastname: Wu
authors.forename: Melissa
authors.initials: M
authors.lastname: Crawford
authors.forename: Wenrui
authors.initials: W
authors.lastname: Duan
authors.forename: Emily E
authors.initials: EE
authors.lastname: Wilding
authors.forename: Li
authors.initials: L
authors.lastname: Gao
authors.forename: S Patrick
authors.initials: SP
authors.lastname: Nana-Sinkam
authors.forename: Miguel A
authors.initials: MA
authors.lastname: Villalona-Calero
authors.forename: Robert A
authors.initials: RA
authors.lastname: Baiocchi
authors.forename: Gregory A
authors.initials: GA
authors.lastname: Otterson
mesh_terms: D000818:Animals; D002417:Cattle; D002460:Cell Line; D004279:DNA, Viral / Q000032:analysis; D005404:Fixatives; D005557:Formaldehyde; D004854:Herpesvirus 4, Human; D006801:Humans; D007150:Immunohistochemistry / Q000379:methods*; D017403:In Situ Hybridization / Q000379:methods*; D035683:MicroRNAs / Q000032:analysis; D009693:Nucleic Acid Hybridization; D027383:Papillomaviridae; D016612:Paraffin Embedding; D016133:Polymerase Chain Reaction; D011108:Polymers; D011309:Preservation, Biological; D012313:RNA; D016707:Tissue Fixation / Q000379:methods
chemical_list: D004279:DNA, Viral; D005404:Fixatives; D035683:MicroRNAs; D011108:Polymers; D005557:Formaldehyde; D012313:RNA; C003043:paraform