The esiRNA technology is a powerful tool for loss of function gene studies. To illustrate the potency of silencing triggers quantitative real time (qRT)-PCR is often used to measure the knock-down rates. The knock-down validation on mRNA level was here performed 24 hours post transfection of esiRNA using qRT-PCR. To be able to assess knock-down rates, the expression levels of the mRNA of interest was compared to cells (HeLa or mouse ES) simultaneously transfected with Renilla Luciferase (negative control).
However, a more valuable measure of the knock-down potency in an RNAi experiment is the reduction in protein level. We therefore also validate the knock-down rates of esiRNAs on protein level using quantitative western blot analysis (Odyssey, Li-COR). The time point for maximum knock-down rate for each protein can vary significantly, as it is depending on factors such as protein-stability, turn-over rate or cell proliferation rates. The knock-down validation on protein level was here performed at 72 hours post transfection of esiRNA in HeLa cells. To be able to assess knock-down rates, the expression levels of the protein of interest was compared to HeLa cells simultaneously transfected with Renilla Luciferase (negative control).
Two approaches were used to monitor knock-down at protein level: 1. Specific antibodies for the protein of interest were used for the quantitative western blot analysis. 2. Proteins of interest were GFP-tagged on a bacterial artificial chromosome (BAC) and stably integrated into the genome of HeLa cells, allowing for near physiological expression (Poser I. et al Nat Methods. 2008 May;5(5):409-15). Using a GFP antibody the detection by quantitative western blot analysis of the protein of interest is straightforward.
* Average of 3 biological replicates
|esiRNA ID||Gene Name||Gene Description||Ensembl ID||RefSeq ID||Knock-Down Rate|
|EMU153941||Ncl||nucleolin||ENSMUSG00000026234||92,78 ± 2,74|
|EMU055581||Wdr5||WD repeat domain 5||ENSMUSG00000026917||NM_080848||89,18 ± 1,65|
|EMU013201||Smc1a||structural maintenance of chromosomes 1A||ENSMUSG00000041133||NM_019710||88,73 ± 4,24|
|EMU016021||Smad4||MAD homolog 4 (Drosophila)||ENSMUSG00000024515||NM_008540||85,97 ± 0,09|
|EMU012081||Apc||adenomatosis polyposis coli||ENSMUSG00000005871||NM_007462||85,5 ± 1,08|
|EMU043301||Rad21||RAD21 homolog (S. pombe)||ENSMUSG00000022314||NM_009009||82,38 ± 3,01|
|EMU084001||Cnot1||CCR4-NOT transcription complex, subunit 1||ENSMUSG00000036550||NM_153164|
|78,78 ± 7,03|
|EMU081351||Pou5f1||POU domain, class 5, transcription factor 1||ENSMUSG00000024406||NM_013633||78,67 ± 10,8|
|EMU045041||Cpsf3||cleavage and polyadenylation specificity factor 3||ENSMUSG00000054309||NM_018813||77,33 ± 1,1|
|EMU031881||Ctr9||Ctr9, Paf1||ENSMUSG00000005609||NM_009431||76,67 ± 4,6|
|EMU167331||Leo1||Leo1, Paf1||ENSMUSG00000042487||75,91 ± 6,87|
|EMU183581||Nanog||Nanog homeobox||ENSMUSG00000012396||75,66 ± 7,98|
|EMU088331||Rtf1||Rtf1, Paf1||ENSMUSG00000027304||NM_030112||74,38 ± 6,39|
|EMU079171||Adam19||a disintegrin and metallopeptidase domain 19 (meltrin beta)||ENSMUSG00000011256||NM_009616||72,12 ± 6,33|