The search for an optimal DNA, RNA, and protein detection by in situ hybridization, immunohistochemistry, and solution-based methods

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• 作者：Fengting Yan ; Xin Wu ; Melissa Crawford ; Wenrui Duan ; Emily E. Wilding ; Li Gao ; S. Patrick Nana-Sinkam ; Miguel A. Villalona-Calero ; Robert A. Baiocchi ; Gregory A. Otterson
• 关键词：In situ hybridization ; Immunohistochemistry ; Fixation ; microRNA ; Real-time PCR ; EBV ; Papillomavirus
• 刊名：Methods
• 出版年：2010
• 出版时间：December 2010
• 年：2010
• 卷：52
• 期：4
• 页码：281-286
• 全文大小：1073 K

文摘

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.