Uhlmann V, Rolfs A, Mix E, Silva I, Hully J, Lu L, Lohman K, Howells D, Picton S, O'Leary J J
Department of Pathology, Cornell University Medical College, New York Hospital, NY 10021, USA.
Mol Pathol. 1998 Jun;51(3):160-3. doi: 10.1136/mp.51.3.160.
Growing interest now focuses on improvements of in situ polymerase chain reaction (PCR) technology for the detection of DNA and RNA cellular sequences. In this study, reverse transcription PCR in situ hybridisation (RT PCR-ISH) was developed and used to determine gene expression of pyruvate dehydrogenase in a cell model system, using human peripheral blood lymphocytes (PBLs). The success of in cell RNA amplification depends on the type of cell/tissue fixation, cell permeabilisation, and the efficiency of reverse transcription and cDNA amplification. This paper presents new approaches to overcome the critical aspects of fixation, permeabilisation, and reverse transcription when performing in cell RNA amplification. A novel fixative, "Permeafix", possessing fixative and permeabilisation properties, was used for cell fixation procedures. "Permeafix" obviated the need for pre-amplification proteolysis, facilitating entry of PCR reagents to target sequences within the cell. In addition, a simple on step RNA in cell amplification protocol using recombinant Thermus thermophilus (rTth) DNA polymerase, which reverse transcribes mRNA efficiently to cDNA and then catalyses cDNA amplification, was used. The value of a semi-junctional primer system for in cell gene expression studies, without the need to perform DNase digestion, is demonstrated.
目前，人们越来越关注改进用于检测DNA和RNA细胞序列的原位聚合酶链反应（PCR）技术。在本研究中，开发了逆转录PCR原位杂交（RT PCR-ISH）并将其用于确定细胞模型系统中丙酮酸脱氢酶的基因表达，该细胞模型系统使用人外周血淋巴细胞（PBL）。细胞内RNA扩增的成功取决于细胞/组织固定的类型、细胞通透化以及逆转录和cDNA扩增的效率。本文介绍了在进行细胞内RNA扩增时克服固定、通透化和逆转录关键环节的新方法。一种具有固定和通透化特性的新型固定剂“Permeafix”用于细胞固定程序。“Permeafix”无需预扩增蛋白酶解，便于PCR试剂进入细胞内的靶序列。此外，还使用了一种简单的一步法细胞内RNA扩增方案，该方案使用重组嗜热栖热菌（rTth）DNA聚合酶，其能有效地将mRNA逆转录为cDNA，然后催化cDNA扩增。证明了半连接引物系统在细胞内基因表达研究中的价值，无需进行DNase消化。
