Power sybr green cells to ct kit

IDT TriFECTa™ 27-mer duplexes (three duplexes per target)
HSC.RNAI.N00250.12 (NEUROD1), HSC.RNAI.N006160.12 (NEUROD2), HSC.RNAI.N000280.12 (PAX6),
HSC.RNAI.N005604.12 (POU3F2) and HSC.RNAI.N001128128.12 (ZEB1) were used according to the
manufacturer guidelines including the TYE-563-DS-transfection control (IDT,
TriFECTa™ kit) and the negative control NC1 Control Duplex (IDT,
TriFECTa™ kit). In total, 50 nM siRNA duplexes (1/4 of each duplex +
1/4 TYE-563-DS-transfection control for single siRNA targets and 1/8 of each duplex for two targets
+ 1/4 TYE-563-DS-transfection control) were transfected per 96-well plate (containing 10,000
iNGN cells plated 1 day prior to siRNA transfection) using the DharmaFECT siRNA transfection kit
(T-2001–02, Thermo Scientific) according to the user manual (0.5 μl of DharmaFECT
reagent per transfection). The transfections were performed in independent biological triplicates
and related to iNGN cells transfected with 50 nM (3/4 negative control NC1 Control Duplex and 1/4
TYE-563-DS-transfection control). After 24 h, the transfections were monitored for the fluorescent
TYE-563 probes and the doxycycline induction was started. Cell samples were harvested 1 and 3 days
post doxycycline induction using the Power SYBR® Green Cells-to-CT™ Kit (4402953,
Ambion).

Lysis of the worm pellets was performed with 5 µl of worm pellet and 45 µl of the lysis solution from the Power SYBR green Cells-to-CT kit (Ambion). Ten freeze-thaw cycles and 30 min shaking at room temperature were performed before the lysis incubation step. The reverse transcription-quantitative PCR (RT-qPCR) was performed according to the manufacturer’s instructions. The qPCR was run on a Step One Plus real-time PCR system (Applied Biosystems). The analysis was done using the ΔΔC_T method. The threshold cycle (C_T) values of four to six biological replicates per experiment were pooled to generate the average ΔC_T for each strain and an associated standard error (SD). The ΔΔC_T value was calculated with ΔC_T in strain N2 as the calibrator. The 2^−ΔΔCT was plotted as the fold change, and the interval of confidence is represented by error bars as 2^{−ΔΔCT + SD(ΔCT)} and 2^{−ΔΔCT − SD(ΔCT)}. The significance of the differences observed was tested by a two-tailed Mann-Whitney U test at a significance level of P < 0.01. The experiments were repeated independently at least twice, and one representative example is shown in the figures.

Real‐time RT–PCR using the PowerSYBR^® Green Cells‐to‐CT™ kit (Ambion) was performed after puromycin selection in order to determine Brca1 mRNA levels. Reverse transcriptase reactions were performed in a Veriti^® 96‐Well Thermal Cycler (Applied Biosystems, Life Technologies Corporation). PCRs were performed using a StepOnePlusTM Real‐Time PCR System (Applied Biosystems, Life Technologies Corporation). The primers used to amplify mouse Brca1 mRNA: ATG CTC TGG CAG CAT GTT CT and CAC TCT GCG AGC AGT CTT CA. Primers for mouse β‐actin (GCT CTG GCT CCT AGC ACC AT and CCA CCG ATC CAC ACA GAG TAC) were used as an endogenous control. All qPCRs were performed in triplicate.

Real-time RT–PCR using the PowerSYBR® Green Cells-to-CT™ kit (Ambion) was performed after puromycin selection in order to determine Exo1, Dna2 and Blm mRNA levels. Reverse transcriptase reactions were performed in a Veriti® 96-Well Thermal Cycler (Applied Biosystems, Life Technologies Corporation). PCRs were performed using a StepOnePlus™ Real-Time PCR System (Applied Biosystems, Life Technologies Corporation). The primers used to amplify mouse transcripts were as follows: Exo1 mRNA: ACC AGC TCG TGT TCG ACC CCA and CAC CGA CGT ACT GCC CAG CG, Dna2 mRNA: ACC ACC ATC TGT GCC CTG GTG A and AAC GGC GGA GTG CGT GTA GC and Blm mRNA: TGT CGG CGC GCG GAG TTT and ACA GCA GCC ATG ATC CTC ACT CA. Primers for mouse β-actin (GCT CTG GCT CCT AGC ACC AT and CCA CCG ATC CAC ACA GAG TAC) were used as an endogenous control. All qPCR reactions were performed in triplicate.

Hepatocytes were lysed using the Power SYBR Green Cells-to-C_T kit (Ambion, Grand Island, NY) at the density of 2000 cells/μl. The lysis reaction was stopped with a proportional volume of 10x Stop Solution. Reverse transcription was performed using 22.5 μl of cell lysate in a total volume of 50 μl, and 4 μl of the resulting cDNA were used as template per PCR reaction. PCR amplification used the following primer pairs: Human GAPDH 5′-gcaaattccatggcaccgt-3′/5′-tcgccccacttgattttgg-3′ P. falciparum 18S 5′-tcagataccgtcgtaatctta-3′/5'-aactttctcgcttgcgcgaa-3′ [34 (link)]. Each sample was amplified in triplicate using an Applied Biosystems StepOnePlus and SYBR Green for detection. Thermal cycling proceeded for 2 minutes at 50°C, 10 minutes at 95°C followed by 40 cycles of 15 seconds at 95°C and 1 minute at 60°C. Pf18S copy numbers were calculated using a standard curve generated from serial dilution of a plasmid template containing the Pf18S gene. Samples were normalized to their own GAPDH C_T values.