Fukumura Ryutaro, Takahashi Hirokazu, Saito Toshiyuki, Tsutsumi Yoko, Fujimori Akira, Sato Shinji, Tatsumi Kouichi, Araki Ryoko, Abe Masumi
Transcriptome Profiling Group, National Institute of Radiological Sciences, Anagawa 4-9-1, Inage-ku, Chiba-shi, Chiba 263-8555, Japan.
Nucleic Acids Res. 2003 Aug 15;31(16):e94. doi: 10.1093/nar/gng094.
We have developed an AFLP-based gene expression profiling method called 'high coverage expression profiling' (HiCEP) analysis. By making improvements to the selective PCR technique we have reduced the rate of false positive peaks to approximately 4% and consequently the number of peaks, including overlapping peaks, has been markedly decreased. As a result we can determine the relationship between peaks and original transcripts unequivocally. This will make it practical to prepare a database of all peaks, allowing gene assignment without having to isolate individual peaks. This precise selection also enables us to easily clone peaks of interest and predict the corresponding gene for each peak in some species. The procedure is highly reproducible and sensitive enough to detect even a 1.2-fold difference in gene expression. Most importantly, the low false positive rate enables us to analyze gene expression with wide coverage by means of four instead of six nucleotide recognition site restriction enzymes for fingerprinting mRNAs. Therefore, the method detects 70-80% of all transcripts, including non-coding transcripts, unknown and known genes. Moreover, the method requires no sequence information and so is applicable even to eukaryotes for which there is no genome information available.
我们开发了一种基于扩增片段长度多态性（AFLP）的基因表达谱分析方法，称为“高覆盖度表达谱分析”（HiCEP）。通过对选择性PCR技术进行改进，我们已将假阳性峰的发生率降低至约4%，因此包括重叠峰在内的峰数量显著减少。结果，我们能够明确确定峰与原始转录本之间的关系。这使得建立所有峰的数据库变得切实可行，无需分离单个峰即可进行基因定位。这种精确的筛选还使我们能够轻松克隆感兴趣的峰，并在某些物种中预测每个峰对应的基因。该方法具有高度可重复性，灵敏度足以检测出基因表达中1.2倍的差异。最重要的是，低假阳性率使我们能够通过使用四种而非六种核苷酸识别位点的限制性内切酶对mRNA进行指纹识别，从而广泛覆盖地分析基因表达。因此，该方法可检测到所有转录本的70 - 80%，包括非编码转录本、未知基因和已知基因。此外，该方法不需要序列信息，因此甚至适用于没有可用基因组信息的真核生物。
