esiRNA coding mouse

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 knockdown rates. The knockdown validation on mRNA level was here performed 24 hours post transfection of esiRNA using qRT-PCR. To be able to assess knockdown 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 knockdown rates of esiRNAs on protein level using quantitative western blot analysis (Odyssey, Li-COR). The time point for maximum knockdown rate for each protein can vary significantly, as it is depending on factors such as protein stability, turnover rate or cell proliferation rates. The knockdown validation on protein level was here performed at 72 hours post transfection of esiRNA in HeLa cells. To be able to assess knockdown 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 knockdown 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 IDGene NameGene DescriptionEnsembl IDRefSeq IDKnock-Down RatePDF
EMU153941NclnucleolinENSMUSG0000002623492,78 ± 2,74PDF
EMU055581Wdr5WD repeat domain 5ENSMUSG00000026917NM_08084889,18 ± 1,65 PDF
EMU013201Smc1astructural maintenance of chromosomes 1AENSMUSG00000041133NM_01971088,73 ± 4,24PDF
EMU016021 Smad4 MAD homolog 4 (Drosophila)ENSMUSG00000024515NM_00854085,97 ± 0,09PDF
EMU012081Apcadenomatosis polyposis coliENSMUSG00000005871 NM_00746285,5 ± 1,08PDF
EMU043301Rad21RAD21 homolog (S. pombe)ENSMUSG00000022314NM_00900982,38 ± 3,01PDF
EMU084001Cnot1CCR4-NOT transcription complex, subunit 1ENSMUSG00000036550NM_153164
NM_178078
78,78 ± 7,03PDF
EMU081351Pou5f1POU domain, class 5, transcription factor 1ENSMUSG00000024406NM_01363378,67 ± 10,8PDF
EMU045041Cpsf3cleavage and polyadenylation specificity factor 3ENSMUSG00000054309NM_01881377,33 ± 1,1 PDF
EMU031881Ctr9Ctr9, Paf1ENSMUSG00000005609NM_00943176,67 ± 4,6PDF
EMU167331Leo1Leo1, Paf1ENSMUSG0000004248775,91 ± 6,87PDF
EMU183581NanogNanog homeoboxENSMUSG0000001239675,66 ± 7,98PDF
EMU088331Rtf1Rtf1, Paf1ENSMUSG00000027304 NM_03011274,38 ± 6,39PDF
EMU079171 Adam19a disintegrin and metallopeptidase domain 19 (meltrin beta)ENSMUSG00000011256 NM_00961672,12 ± 6,33PDF