Quantstudio six flex real time pcr system

Total RNA was extracted using TRIzol reagent (Invitrogen) according to the manufacturer’s instructions. cDNA was synthesized through reverse transcription of total RNA using Mir-X miRNA First-Strand Synthesis Kit (Clontech) according to the manufacturer’s instructions. qRT-PCR was performed using PerfeCTa™ SYBR® Green FastMix™ following manufacturer’s instructions. Reaction mix comprised of 5 µL of Green FastMix [2x], 1 µL each of forward miRNA specific primer (10 µM) and mRQ 3′ primer [10 µM], 50 ng of cDNA and MilliQ water to a 10 µL final volume. The PCR program was 95°C for 3 min followed by 40 cycles of 95°C for 30°s and 65°C for 30°s. Amplification specificity was determined by generating dissociation curves. The dissociation program was 95°C for 15°s and 50°C for 15°s, followed by a slow gradient from 50 to 95°C. qRT-PCR reactions were carried out in QuantStudio six Flex Real-Time PCR System (Applied Biosystems). Relative expression was calculated as previously described by Livak and Schmittgen (2001) (link). Small nuclear RNA U6 and 60°S (Glyma.13G318800) genes were used as internal control to normalize gene expression data. Primer sequences used in qRT-PCR assays are provided in Supplementary Table S8.

RNA was isolated from the cells using NucleoSpin RNA kit (Macherey-Nagel) and reverse transcribed to cDNA using iScript cDNA synthesis kit (BioRad). The relative expression of prime editor in the stables compared to the respective wild-type cells was quantified using primers targeting Cas9. GAPDH was used as the normalising internal control. The PCR reaction was carried out using SsoFast EvaGreen Supermixes (Bio-Rad) in QuantStudio six Flex Real-Time PCR System (Applied Biosystems) after three fold dilution of cDNA samples as reported previously (Ravi et al., 2022 (link)).

Total RNA was isolated from all cells using TRIzol reagent (Thermofisher, #15596026) and 100 to 500 ng of RNA was reverse transcribed using the high-capacity cDNA reverse transcription kit (Thermofisher, #4368814). Real time qPCR was done using Fast SYBR Green Master Mix (Applied Biosystems #43–856-12) and ran on QuantStudio six Flex real-time PCR system (Applied Biosystems # 4485691). Individual gene expression levels were calculated using the change in cycling threshold (ΔC_T) method as 2^−ΔCT, where ΔC_T is [C_T (gene of interest)- C_T (housekeeping gene)]. qPCR primer sequences are included in Table 4.

Total RNA was isolated using the TRIzol^® (TaKaRa Bio, Otsu, Shiga, Japan) method following the manufacturer’s instruction. The concentration and quality of the total RNA was checked by NanoDrop 2000 analyzer (Thermo Scientific, Ltd., Wilmington, DE, United States). cDNA was synthesized using the HiScript II QRT SuperMix for qPCR (+gDNA wiper) (Vazyme biotech). qPCR was performed with the QuantStudio six Flex Real-Time PCR System (Life Technologies, United States) using ChamQ SYBR qPCR Master Mix (Vazyme biotech). All reactions were performed in triplicate. Fold differences in gene expression were calculated with the ΔΔCt method using β-actin as the housekeeping gene. The primer sequences for tested genes were as follows (Table 1).

QIA amp DNA Mini Kit (Qiagen, Venlo, Netherlands) was used to extract total DNA (genomic and mitochondrial DNA) in C3H10T1/2 cells following the instructions. The concentration and quality of DNA were determined using NanoDrop 2000 analyzer (Thermo Scientific, United States). qPCR was performed with the QuantStudio six Flex Real-Time PCR System (Life Technologies, United States) using ChamQ SYBR qPCR Master Mix (Vazyme biotech) to analyze the copy number of mtDNA associated with genomic DNA. The primer sequences of COX one and cyclophilin A are shown in Table 1.