Genetic Engineering Research Center, School of Bioengineering, Chongqing University, Chongqing, People's Republic of China.
PLoS One. 2011 Jan 18;6(1):e15711. doi: 10.1371/journal.pone.0015711.
We established a novel method, Gene Expression Profiling via Multigene Concatemers (MgC-GEP), to study multigene expression patterns simultaneously. This method consists of the following steps: (1) cDNA was obtained using specific reverse primers containing an adaptor. (2) During the initial 1-3 cycles of polymerase chain reaction (PCR), the products containing universal adaptors with digestion sites at both termini were amplified using specific forward and reverse primers containing the adaptors. (3) In the subsequent 4-28 cycles, the universal adaptors were used as primers to yield products. (4) The products were digested and ligated to produce concatemers. (5) The concatemers were cloned into the vector and sequenced. Then, the occurrence of each gene tag was determined. To validate MgC-GEP, we analyzed 20 genes in Saccharomyces cerevisiae induced by weak acid using MgC-GEP combined with real-time reverse transcription (RT)-PCR. Compared with the results of real-time RT-PCR and the previous reports of microarray analysis, MgC-GEP can precisely determine the transcript levels of multigenes simultaneously. Importantly, MgC-GEP is a cost effective strategy that can be widely used in most laboratories without specific equipment. MgC-GEP is a potentially powerful tool for multigene expression profiling, particularly for moderate-throughput analysis.
我们建立了一种新的方法，即通过多基因串联（MgC-GEP）进行基因表达谱分析，以同时研究多基因表达模式。该方法包括以下步骤：（1）使用包含接头的特定反转录引物获得 cDNA。（2）在聚合酶链反应（PCR）的最初 1-3 个循环中，使用包含通用接头且两端都具有消化位点的引物扩增包含通用接头的产物。（3）在随后的 4-28 个循环中，使用通用接头作为引物来产生产物。（4）将产物消化并连接以产生串联物。（5）将串联物克隆到载体中并进行测序。然后，确定每个基因标签的出现。为了验证 MgC-GEP，我们使用 MgC-GEP 结合实时反转录（RT）-PCR 分析了弱酸性诱导的酿酒酵母中的 20 个基因。与实时 RT-PCR 的结果和微阵列分析的先前报告相比，MgC-GEP 可以精确地同时确定多基因的转录水平。重要的是，MgC-GEP 是一种具有成本效益的策略，可以在没有特定设备的情况下广泛应用于大多数实验室。MgC-GEP 是一种用于多基因表达谱分析的潜在强大工具，特别适用于中等通量分析。
