With specific Antibodies

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 IDGene NameGene DescriptionEnsembl IDRefSeq IDKnock-Down RateCatalog Number (Sigma-Antibody)

Pic / PDF

EHU083501AKT1v-akt murine thymoma viral oncogene homolog 1ENSG00000142208

NM_001014432
NM_001014431
NM_005163

88%P2482-.2MLPic / PDF
EMU081351Pou5f1POU domain, class 5, transcription factor 1ENSMUSG00000024406NM_01363385%P0082-200ULPic / PDF
EHU140181PAK1p21 protein (Cdc42/Rac)-activated kinase 1ENSG00000149269 up to 84%P3237-.2MLPic / PDF
EHU048671CATcatalaseENSG00000121691NM_00175284%C0979-.2MLPic / PDF
EHU106441PTENphosphatase and tensin homologENSG00000171862NM_00031483%P3487Pic / PDF
EHU114671HSP90AA1heat shock protein 90kDa alpha (cytosolic), class A member 1ENSG00000080824

NM_005348
NM_001017963

83%H1775-.2MLPic / PDF
EHU051011PLK1polo-like kinase 1ENSG00000166851NM_00503081%P5998-200ULPic / PDF
EHU114431KRASv-Ki-ras2 Kirsten rat sarcoma viral oncogene homologENSG00000133703

NM_004985
NM_033360

77%R3400-.1MGPic / PDF
EHU135281BCL2B-cell CLL/lymphoma 2ENSG00000171791 76%B3170-.2MLPic / PDF
EHU032581CAPN1calpain 1, (mu/I) large subunitENSG00000014216 76%C267-100ULPic / PDF
EHU143271ILKintegrin-linked kinaseENSG00000166333 up to 76% I0783-200UL Pic / PDF
EHU031251CAV1caveolin 1, caveolae protein, 22kDaENSG00000105974NM_00175374%SAB4200216Pic / PDF
EHU072971MAPK8mitogen-activated protein kinase 8ENSG00000107643

NM_139046
NM_002750
NM_139047
NM_139049

70%SAB4200176-200ULPic / PDF
EHU070511CDK4cyclin-dependent kinase 4ENSG00000135446NM_00007570%C8218-.5MLPic / PDF
EHU123221TP53tumor protein p53ENSG00000141510

NM_000546
NM_001126112
NM_001126113
NM_001126114
NM_001126115
NM_001126116
NM_001126117

68%P9124-200ULPic / PDF
EHU141291ABL1c-abl oncogene 1, non-receptor tyrosine kinaseENSG00000097007 63%A5844-.2MLPic / PDF
EHU146741GAPDHglyceraldehyde-3-phosphate dehydrogenaseENSG00000111640 63%G8795-200ULPic / PDF
EHU088101VCLvinculinENSG00000035403

NM_014000
NM_003373

51%SAB4200080-200ULPic / PDF
EHU060231FASFas (TNF receptor superfamily, member 6)ENSG00000026103

NM_152871
NM_152874
NM_152873
NM_152877
NM_152875
NM_152876
NM_152872
NM_000043

45%F4424-.1MLPic / PDF
EHU139421CTNNB1catenin (cadherin-associated protein), beta 1, 88kDaENSG00000168036 44%C7082-.2MLPic / PDF

AKT1 / v-akt murine thymoma viral oncogene homolog 1

CAT / catalase

PLK1 / polo-like kinase 1

CAPN1 / calpain 1, (mu/I) large subunit

MAPK8 / mitogen-activated protein kinase 8

ABL1 / c-abl oncogene 1, non-receptor tyrosine kinase

FAS / Fas (TNF receptor superfamily, member 6)

Pou5f1 / POU domain, class 5, transcription factor 1

PTEN / phosphatase and tensin homolog

KRAS / v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog

ILK / integrin-linked kinase

CDK4 / cyclin-dependent kinase 4

GAPDH / glyceraldehyde-3-phosphate dehydrogenase

CTNNB1 / catenin (cadherin-associated protein), beta 1, 88kDa

PAK1 / p21 protein (Cdc42/Rac)-activated kinase 1

HSP90AA1 / heat shock protein 90kDa alpha (cytosolic), class A member 1

BCL2 / B-cell CLL/lymphoma 2

CAV1 / caveolin 1, caveolae protein, 22kDa

TP53 / tumor protein p53

VCL / vinculin