Lightcycler 480 sybr green 1 mix

In both human liver tissue and human cell lines, RNA was isolated using the RNeasy Mini Kit (QIAGEN). QuantiTect Reverse Transcription Kit (QIAGEN) was used to synthesize complementary DNA. Quantitative real time PCR was prepared with LightCycler 480 SYBR®Green I Mix (Roche) and performed with the LightCycler 480 (Roche). As an endogenous control for mRNA levels expression was normalized against hypoxanthine-guanine phosphoribosyl transferase (HPRT). The Light Cycler® 480 SW 1.5 software was used to analyse the data. Primers were purchased from Metabion.

RT-PCR was used to evaluate the efficacy of sp.-MOs. cDNA from 24 hpf, forward primer (5′- ATCAAGCCGGAGCCAGAGGAGGTG-3′) and reverse primer (5′-GCCGTCGGTGAAGTGCCAGGTG-3′) were used. RT-qPCR [39 (link)] was used to compare expression levels of klf3, klf12a or klf12b in wild type and homozygous F5 klf8^d25 and klf8ⁱ¹⁷ mutant embryos. cDNA was generated by a GoScript Reverse transcription system (Promega) using total RNA isolated from 10 to 12 s stage wild type or two homozygous klf8^d25 and klf8ⁱ¹⁷ mutant embryos. RT-qPCR was conducted in a 10 μL reaction containing 1× LightCycler 480 SYBR Green I Mix (Roche), respective primer pairs (5 μg) and 1/10 cDNA from wild type or mutant embryos. PCR conditions were 95 °C for 10 min, then 55 cycles of, 95 °C for 10 s, 60 °C for 10 s, and 72 °C for 10 s, followed by a 4 °C pause. Primer pairs for klf3 were forward (5′-TATCCAAGTGGACATTACTGTGGG-3′) and reverse (5′-CAGTGGGCAACACAGAACGGCAG-3′). Primer pairs for klf12a were forward (5′-GAGCGGGTCTCTTTCTGCCAGTG-3′) and reverse (5′-CAATAAACCGTATGAGGGAAAGGC-3′). Primer pairs for klf12b were forward (5′-GGCAATCCCTGCTCCTCAGAAAC-3′) and reverse (5′-CCACATCGTAGACTCCAAAATGCG-3′).

RNA samples from cells were obtained using the RNeasy kit (Cat no. 74134 Qiagen). according to the manufacturer’s instructions. cDNA was synthesized using QuantiTect Reverse Transcription kit (Cat no. 205314 Qiagen). Real-time quantitative RT-PCR on cDNAs was carried out with the LightCycler 480 SYBR Green I mix (Cat no. 04887352001 Roche) using the Light Cycler 480 II detection system (Roche) with the following conditions: 95 °C, 5 min (95 °C, 10 s; 60 °C, 10 s; 72 °C, 15 s) × 40. Fold change values were calculated using the ΔΔCt method. In brief, an internal control (HPRT1) was used as a ‘normalizer’ to calculate the ΔCt value. Next, the ΔΔCt value was calculated between the ‘control’ group and the ‘experimental’ group. Finally, the fold change was calculated using 2(−ΔΔCt). Biological replicates were grouped in the calculation of the fold-change values. For TFEB targets gene analysis HeLa FLCN KO cells were co-transfected with either RagD mutants constructs and EGFP empty vector using the Fugene reagent (cat no. E2312 Promega) following the manufacturer’s instructions. 48 h upon transfection, GFP-positive cells were sorted via FACS and RNA was extracted for the subsequent analysis.