Identification of differentially expressed genes from limited amounts of RNA.
Identification of differentially expressed genes from limited amounts of RNA.
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从有限数量的 RNA 中鉴定差异表达基因。
DOI：
10.1093/nar/28.7.e27
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发表时间：
2000
影响因子：
14.9
通讯作者：
Pardee,AB
中科院分区：
文献类型：
--
作者：
Bosch,I;Melichar,H;Pardee,AB
通过差异显示（DD）鉴定细胞RNA表达谱涉及来自RNA的RT-PCR产物的可视化。传统上，DD方案对于每个RT反应需要200-500 ng RNA。因此，DD的限制因素是可用RNA的量和RT反应的灵敏度。通过在我们的方法中替换逆转录酶的类型，DD的灵敏度提高了100倍。非常重要的是，获得的cDNA种类的分子量更高，增加了检测具有较少背景条带的差异显示基因的机会。由于在PCR反应步骤中利用Taq聚合酶抗体促进DNA合成，假阳性和背景一般也降低。这里描述的逆转录酶可能具有更大的引发能力以及强的持续合成能力，这将解释与更标准的逆转录酶相比实现的更高的灵敏度。此外，当RNA的量有限时，强烈建议对样品应用更灵敏的DD。
The identification of cellular RNA expression profiles by differential display (DD) involves the visualization of RT–PCR products from the RNA. Traditionally, DD protocols require 200–500 ng RNA for each RT reaction. Thus, the limiting factor in DD is the amount of RNA available and the sensitivity of the RT reaction. By replacing the type of reverse transcriptase in our method, the sensitivity of DD increased up to 100-fold. Very significantly, the cDNA species obtained are higher in molecular weight, increasing the chances of detection of differential display genes with less background bands. The false positives and background in general also decreased due to the utilization ofTaqpolymerase antibody to facilitated DNA synthesis in the PCR reaction step. The reverse transcriptases described here may have a greater priming capacity as well as strong processivity which would explain the higher sensitivity accomplished in comparison to more standard reverse transcriptases. Additionally, the application of a more sensitive DD to samples when the amount of RNA is limited would be highly recommended.