Cells were plated at 105 cells/well, allowed to adhere overnight and then transfected with 50 nM SMARTpool ON-TARGETplus VHL siRNA (Dharmacon) or ON-TARGETplus GAPD Control Pool (Dharmacon) using DharmaFECT 1 Transfection Reagent (Dharmacon). Culture media was replaced 8 h post-transfection and cells were assayed by western blotting 48 h or 72 h post-transfection.
On targetplus gapd control pool
Manufactured by Horizon Discovery
The ON-TARGETplus GAPD Control Pool is a laboratory product designed for use as a positive control in gene expression and gene knockdown studies. It consists of a pool of small interfering RNAs (siRNAs) targeting the glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene, which is commonly used as a reference gene in such experiments.
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Lab products found in correlation
4 protocols using on targetplus gapd control pool
1
VHL Knockdown in Cell Lines
2
FOXO1 Knockdown in MCF7 Cells
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ON-TARGETplus SMARTpool FOXO1 siRNA and ON-TARGETplus GAPD Control Pool (Dharmacon; Thermo Scientific) were used at a final concentration of 100 nM. MCF7 cells were reverse transfected in a 6-well plate using Lipofectamine RNAiMAX reagent (Invitrogen) per the manufacturer’s protocol.
3
FOXO1 Knockdown in MCF7 Cells
4
Quantitative Real-Time RT-PCR for Rabbit FGF19 Gene Expression
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Isolation of total RNA from tissues and cells, cDNA synthesis and quantitative real-time RT-PCR were performed as previously described (Comeglio et al. 2017) (link). Specific PCR primers for rabbit target genes were designed on sequences available at NCBI GenBank (http://www.ncbi.nlm.nih. gov) or Ensemble Genome (http://www.ensembl.org) and were purchased from Life Technologies. In particular, rabbit FGF19 sequence (ENSOCUT00000003867.3) was used as template. FGF15 and FGF19 are orthologous proteins with similar functions, with the former described in the mouse and the latter found in the rabbit (Shang et al. 2014) (link). The 18S ribosomal RNA subunit was used as the reference gene for the relative quantization of the target genes based on the comparative threshold cycle (Ct) 2 -ΔΔCt method (Livak & Schmittgen 2001) (link).
siRNA targeting human FXR (NR1H4 ON-TARGETplus SMARTpool) and TGR5 (GPBAR1 ON-TARGETplus SMARTpool), positive (ON-TARGETplus GAPD Control Pool) and negative (ON-TARGETplus Non-Targeting Pool) sequences were purchased from Dharmacon (Lafayette, CO,
siRNA targeting human FXR (NR1H4 ON-TARGETplus SMARTpool) and TGR5 (GPBAR1 ON-TARGETplus SMARTpool), positive (ON-TARGETplus GAPD Control Pool) and negative (ON-TARGETplus Non-Targeting Pool) sequences were purchased from Dharmacon (Lafayette, CO,
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