Quantstudio 6 flex real time pcr system thermocycler

For human peripheral blood samples, we used TRIzol method or RNeasy Mini Kit (Qiagen, Germantown, MD, USA) following the manufacturer’s conditions to obtain total RNA. Reverse transcription was performed with the High-Capacity cDNA Reverse Transcription kit (Thermo Fisher, Madrid, Spain), following the manufacturer’s instructions. RT-PCR reactions were performed as previously described [46 (link)] using QuantStudio 12 K Flex Real-Time PCR System (Life Technologies Inc., Carlsbad, CA, USA), with specific Taqman probes: FNIP2 (Hs01574322_m1), FLCN (Hs00376065_m1), FNIP1 (Hs00382846_m1), GAPDH (Hs02786624_g1). The 2^−ΔΔCt method was applied to calculate the relative gene expression. COSMIC Cell Lines Project (https://cancer.sanger.ac.uk/cell_lines) was used to download genotypes and gene expression values for FNIP2 in 965 cell lines.
For mouse tissues and primary cells, RNA was extracted using TRIzol together with RNeasy Mini Kit (Qiagen, 74106) or Direct-zol RNA Miniprep (Zymo Research, R2051). Reverse transcription was performed using SuperScript IV VILO Master Mix (Invitrogen, 11756500). Quantitative real-time PCR was run in triplicates using GoTaq qPCR Master Mix (Promega, A6001) in a QuantStudio 6 Flex Real-Time PCR System thermocycler (Applied Biosystems). The 2^−ΔΔCt method was applied to calculate the relative gene expression, using β-actin as reference gene.

Total RNA was extracted from tissues or cells using Trizol (Invitrogen #15596026) according to manufacturer’s instructions. For RNA extraction from tissue, RNeasy Tissue Kit (Qiagen #74106) was used. For RNA extraction from cells, Direct-zol RNA Minipreps Kits (ZYMO RESEARCH #R2050) were used. Samples were retro-transcribed using SuperScript IV VILO Master Mix (Invitrogen #11756500) following manufacturer’s instructions. Quantitative real-time PCR was run in triplicates using GoTaq qPCR Master Mix (Promega #A6001) with a reaction volume of 10 μL in a QuantStudio 6 Flex Real-Time PCR System thermocycler (Applied Biosystems). Data were analyzed by the change-in-threshold (2^−ΔΔCT) method, using β-actin as “housekeeping” reference gene for mouse samples and Atp5f1 for pig samples. Results are represented as fold changes relative to the mean expression levels of the control condition. Primer sequences are listed in Supplementary Data 2.