[生物][实验基础]siRNA/miRNA转染:目的基因为何不降反升？

dr.qinan

很多做过siRNA/miRNA的人，也许在实验中发现，siRNA/miRNA转染细胞后，目的基因表达不仅没有下调，反而上升！很多人都认为不可思议，更多的时候把这种现象归咎于自己实验操作时的失误，或者甚至放弃进一步探讨。最近的nature biotechnology的一篇paper指出，这种现象是存在的，其原因可能在于细胞内外源性的miRNA与小RNA分子竞争性结合某些细胞内装置，从而引起一种“饱和效应”。这篇文章解决了许多人的困惑，同时也对合理设计siRNA/miRNA不无启示。

Nat Biotechnol. 2009 May 24. AOP
Transfection of small RNAs globally perturbs gene regulation by endogenous microRNAs.

Transfection of small RNAs (such as small interfering RNAs (siRNAs) and microRNAs (miRNAs)) into cells typically lowers expression of many genes. Unexpectedly, increased expression of genes also occurs. We investigated whether this upregulation results from a saturation effect-that is, competition among the transfected small RNAs and the endogenous pool of miRNAs for the intracellular machinery that processes small RNAs. To test this hypothesis, we analyzed genome-wide transcript responses from 151 published transfection experiments in seven different human cell types. We show that targets of endogenous miRNAs are expressed at significantly higher levels after transfection, consistent with impaired effectiveness of endogenous miRNA repression. This effect exhibited concentration and temporal dependence. Notably, the profile of endogenous miRNAs can be largely inferred by correlating miRNA sites with gene expression changes after transfections. The competition and saturation effects have practical implications for miRNA target prediction, the design of siRNA and short hairpin RNA (shRNA) genomic screens and siRNA therapeutics.

shenww

我认为这篇文章揭示了一个真正做科研的态度

joseay963

严谨啊