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 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 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