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.
|esiRNA ID||Gene Name||Gene Description||Ensembl ID||RefSeq ID||Knock-Down Rate||Pic / PDF|
|EMU072781||Atg12||autophagy-related 12 (yeast)||ENSMUSG00000032905||NM_026217||91%||Pic / PDF|
|EMU052231||Park7||Parkinson disease (autosomal recessive, early onset) 7||ENSMUSG00000028964||NM_020569||89%||Pic / PDF|
|EMU014921||Parp1||poly (ADP-ribose) polymerase family, member 1||ENSMUSG00000026496||NM_007415||87%||Pic / PDF|
|EMU014271||Smad3||MAD homolog 3 (Drosophila)||ENSMUSG00000032402||NM_016769||84%||Pic / PDF|
|EMU047871||Akt2||thymoma viral proto-oncogene 2||ENSMUSG00000004056|
|82%||Pic / PDF|
|EMU001711||Lyplal1||lysophospholipase-like 1||ENSMUSG00000039246||NM_146106||82%||Pic / PDF|
|EMU044861||Cdt1||chromatin licensing and DNA replication factor 1||ENSMUSG00000006585||NM_026014||81%||Pic / PDF|
|EMU190491||Prkaa1||protein kinase, AMP-activated, alpha 1 catalytic subunit||ENSMUSG00000050697||74%||Pic / PDF|