Fam mgb taqman gene expression assays

Total RNA was isolated with Trizol reagent (Invitrogen) following RNA purification using the RNeasy Mini Kit (Qiagen) and converted to cDNA by High Capacity RNA-to-cDNA Kit (Applied Biosystems), according to the manufacturer's protocols. An assay comprising known human and murine CLEC16A RNA transcripts as well as control genes (β-Actin and HRPT1) were measured by real time PCR on a ViiA™ 7 Real Time PCR System, using predesigned 20X FAM-MGB TaqMan gene expression assays available from Applied Biosystems. All assays had primers covering exon-exon boarders to avoid DNA contamination. Triplicates were used for all samples included in the experiment. All PCR runs were performed on ViiA™ 7 Real Time PCR System using ViiA7 RUO software v1.2.2 (Life Technologies).

As described in our previous publication^{37 (link)}, total RNA was isolated with Trizol reagent (Invitrogen) following RNA purification using the RNeasy Mini Kit (Qiagen) and converted to cDNA by High Capacity RNA-to-cDNA Kit (Applied Biosystems), according to the manufacturer’s protocols. Assay comprising known human and murine CLEC16A RNA transcripts as well as control genes (β-actin and HRPT1) were measured by real time PCR on a ViiA 7 Real Time PCR System using predesigned 20X FAM-MGB TaqMan gene expression assays available from Applied Biosystems. All assays had primers covering exon-exon boarders to avoid DNA contamination. Triplicates were used for all samples included in the experiment. All PCR runs were performed on ViiA 7 Real Time PCR System using ViiA7 RUO software v1.2.2 (Life Technologies).

A detailed protocol for confirming gene knockdown with RT-PCR can be found at https://doi.org/10.17504/protocols.io.dm6gp3yo1vzp/v1. Relative gene expression quantifications were performed using qRT-PCR on a 7900HT Fast Real-Time PCR System (Applied Biosystem; RRID:SCR_018060), using VIC-MGB human ACTB as an endogenous control (β-actin, ThermoFisher #4326315E) and FAM-MGB TaqMan Gene Expression Assays (ThermoFisher, assay IDs: Hs00204417_m1-NDUFA8, Hs00244980_m1-NDUFA1, Hs00984333_m1-NDUFA12, Hs00958815_g1-NDUFB7, Hs00192290_m1-NDUFAB1, Hs00853558_g1-NDUFB4, Hs00159597_m1-NDUFS8, and Hs02578754_g1-NDUFS5). cDNA and PCR reactions were prepared using the Cells-to-CT kit (ThermoFisher), following their protocol with a standard reverse transcription cycle (37 °C for 6 min, followed by 95 °C for 5 min and holding at 4 °C), and standard qRT-PCR conditions (UDG incubation of 50 °C for 2 min, enzyme activation at 95 °C for 10 min, and PCR cycles of 95 °C for 15 s, 60 °C for 1 min, repeated for 40 cycles). All reactions were performed in a 384-well plate format in duplicate and compiled from at least two independent experiments. Fold changes in expression were calculated using the 2^−ΔΔCT method.