Ambion sirna

Custom designed Ambion siRNA targeting the optimized-NTSR1 sequence were ordered from thermofisher with the following sequence:

5′-TACGTGAGTTCCACCATAAAT

5′-ACTGTCTGTGGAAAGATATTT

5′-AGCGTGCTGAACACCATTATA

HEK293T-NTSR1 cells were transfected with the Lipofectamine RNAimax (thermofisher) following the manufacturer recommendations.

Lipid mediated siRNA transfection was used to knock down arntl expression. For anrtl silencing, pooled arntl Qiagen flexitube siRNAs (catalog # 1027416) were transfected into KO MEFs using Thermo Fisher RNAiMAX reagent with transfection performed per manufacturer instructions. Thermo Fisher Ambion siRNA (catalog # AM16708) was used for ribose-5-phosphate isomerase (RPIA) silencing. Arntl was overexpressed in WT MEFs using an Origene pCMV6 vector plasmid (catalog # MR209553) using Thermo Fisher Lipofectamine 3000 reagent. RNA was harvested 24 h post transfection.

Gene knockdown experiments were conducted in iCell-CMs using commercially available small interfering (si) RNAs (Thermo Fisher Scientific) targeting the identified candidate genes. hiPSC-CMs were plated in 96-well dishes at an approximate density of 1.71 × 10⁴ cells/well. Transfections were performed in triplicate using Invitrogen’s Lipofectamine® RNAiMAX reagent (Thermo Fisher Scientific) per manufacturer’s instructions. Each siRNA transfection was conducted using two unique Ambion® siRNAs (Thermo Fisher Scientific) per gene to ensure efficient gene knockdown. Invitrogen’s Block-iT™ fluorescent control (Thermo Fisher Scientific) was used as a positive control, with successful nuclear-localized fluorescence. Two negative controls were run with no discernable effect: (1) the transfection reagent without siRNAs and (2) Ambion® Silencer™ Negative Control No. 3 siRNA (Thermo Fisher Scientific). RNA extractions were performed with Norgen’s Total RNA purification 96-well kit (Norgen Biotek Corp). The knockdown efficiency was determined via real-time quantitative PCR (RT-qPCR) using TaqMan™ (Roche) gene expression assays following the manufacturer’s recommendations, normalizing to GAPDH, and compared to the Silencer Negative Control No. 3.