Czechowski Tomasz, Bari Rajendra P, Stitt Mark, Scheible Wolf-Rüdiger, Udvardi Michael K
Max-Planck Institute of Molecular Plant Physiology, Am Mühlenberg 1, 14476 Golm, Germany.
Plant J. 2004 Apr;38(2):366-79. doi: 10.1111/j.1365-313X.2004.02051.x.
Summary To overcome the detection limits inherent to DNA array-based methods of transcriptome analysis, we developed a real-time reverse transcription (RT)-PCR-based resource for quantitative measurement of transcripts for 1465 Arabidopsis transcription factors (TFs). Using closely spaced gene-specific primer pairs and SYBR Green to monitor amplification of double-stranded DNA (dsDNA), transcript levels of 83% of all target genes could be measured in roots or shoots of young Arabidopsis wild-type plants. Only 4% of reactions produced non-specific PCR products. The amplification efficiency of each PCR was determined from the log slope of SYBR Green fluorescence versus cycle number in the exponential phase, and was used to correct the readout for each primer pair and run. Measurements of transcript abundance were quantitative over six orders of magnitude, with a detection limit equivalent to one transcript molecule in 1000 cells. Transcript levels for different TF genes ranged between 0.001 and 100 copies per cell. Only 13% of TF transcripts were undetectable in these organs. For comparison, 22K Arabidopsis Affymetrix chips detected less than 55% of TF transcripts in the same samples, the range of transcript levels was compressed by a factor more than 100, and the data were less accurate especially in the lower part of the response range. Real-time RT-PCR revealed 35 root-specific and 52 shoot-specific TF genes, most of which have not been identified as organ-specific previously. Finally, many of the TF transcripts detected by RT-PCR are not represented in Arabidopsis EST (expressed sequence tag) or Massively Parallel Signature Sequencing (MPSS) databases. These genes can now be annotated as expressed.
摘要 为克服基于DNA芯片的转录组分析方法固有的检测限，我们开发了一种基于实时逆转录（RT）-PCR的资源，用于定量测量1465个拟南芥转录因子（TF）的转录本。使用紧密间隔的基因特异性引物对和SYBR Green监测双链DNA（dsDNA）的扩增，在拟南芥野生型幼苗的根或芽中可测量所有靶基因中83%的转录水平。只有4%的反应产生非特异性PCR产物。每个PCR的扩增效率由指数期SYBR Green荧光对循环数的对数斜率确定，并用于校正每个引物对每次运行的读数。转录本丰度的测量在六个数量级上是定量的，检测限相当于1000个细胞中的一个转录本分子。不同TF基因的转录水平在每个细胞0.001至100个拷贝之间。在这些器官中只有13%的TF转录本无法检测到。相比之下，22K拟南芥Affymetrix芯片在相同样品中检测到的TF转录本不到55%，转录水平范围被压缩了100倍以上，并且数据在响应范围的下限处尤其不准确。实时RT-PCR揭示了35个根特异性和52个芽特异性TF基因，其中大多数以前未被鉴定为器官特异性。最后，RT-PCR检测到的许多TF转录本在拟南芥EST（表达序列标签）或大规模平行签名测序（MPSS）数据库中未被表征。这些基因现在可以注释为已表达。
