Taniguchi A, Kohsaka H, Carson D A
Department of Medicine, University of California, San Diego, La Jolla 92093-0663.
J Immunol Methods. 1994 Feb 28;169(1):101-9. doi: 10.1016/0022-1759(94)90129-5.
We have developed a non-radioactive method to quantitate precisely levels of gene expression. This method is based on RT-PCR (reverse transcriptase-polymerase chain reaction) with an RNA competitor, followed by the covalent capture of the amplified DNA onto the wells of microtiter plates, and the quantitation of the PCR product by oligonucleotide hybridization and ELISA (enzyme-linked immunosorbent assay). The assay can reproducibly detect 1 zeptomole mRNA. The assay was successfully used to quantitate mRNA levels of the T cell derived cytokines interleukin-2, interleukin-4 and interferon-gamma in resting and stimulated human lymphocytes. Because it is performed in a microtiter ELISA format, this rapid, sensitive and non-radioactive method should facilitate measurements of gene expression, particularly in large clinical studies.
我们开发了一种非放射性方法来精确量化基因表达水平。该方法基于带有RNA竞争物的逆转录聚合酶链反应（RT-PCR），随后将扩增的DNA共价捕获到微孔板孔中，并通过寡核苷酸杂交和酶联免疫吸附测定（ELISA）对PCR产物进行定量。该测定法可重复性地检测到1 zeptomole的mRNA。该测定法已成功用于定量静息和受刺激的人淋巴细胞中T细胞衍生的细胞因子白细胞介素-2、白细胞介素-4和γ干扰素的mRNA水平。由于它以微孔ELISA形式进行，这种快速、灵敏且非放射性的方法应有助于基因表达的测量，特别是在大型临床研究中。
