Transcript imaging with cDNA-AFLP: A step-by-step protocol
Transcript imaging with cDNA-AFLP: A step-by-step protocol
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DOI：
10.1023/a:1007468801806
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发表时间：
1998-06-01
影响因子：
2.1
通讯作者：
Visser, RGF
中科院分区：
文献类型：
--
作者：
Bachem, CWB;Oomen, RJFJ;Visser, RGF
cDNA-AFLP方法允许使用PCR检测差异表达的转录本。本报告提供了一个详细的和更新的cDNA-AFLP程序和分析其各种参数之间的相互作用的协议。我们研究了PCR循环次数和模板稀释度对转录衍生片段（TDFs）数量的影响。我们还研究了使用磁珠合成cDNA和扩增过程中MgCl 2浓度的影响。最后，我们确定了检测水平的cDNA-AFLP方法使用的TDFs的各种大小和组成。我们可以检测TDFs对应于一个单一的拷贝每个细胞的特定转录本的cDNA-AFLP模式，表明高灵敏度的方法。此外，可检测的TDF浓度和片段大小之间没有相关性，强调了扩增反应的高严格性。理论上的考虑和具体应用的方法进行了讨论。
The method of cDNA-AFLP allows detection of differentially expressed transcripts using PCR. This report provides a detailed and updated protocol for the cDNA-AFLP procedure and an analysis of interactions between its various parameters. We studied the effects of PCR cycle number and template dilution level on the number of transcript derived fragments (TDFs). We also examined the use of magnetic beads to synthesise cDNA and the effect of MgCl2 concentration during amplification. Finally, we determined the detection level of the cDNA-AFLP method using TDFs of various sizes and composition. We could detect TDFs corresponding to a single copy per cell of a specific transcript in a cDNA-AFLP pattern, indicating high sensitivity of the method. Also, there was no correlation between concentration of detectable TDF and the fragment size, stressing the high stringency of the amplification reactions. Theoretical considerations and specific applications of the method are discussed.