Rneasy lipid tissue mini protocol kit

Total RNA of hepatic and renal tissues was extracted from 5 to 10 mg of tissue using the RNeasy Lipid Tissue Mini Protocol Kit from Qiagen according to the manufacturer instructions (Qiagen). The obtained RNA was quantified using a Nanodrop 2000 (Thermo scientific). Real-time (RT)-PCR was performed using 1 μg of RNA with the iScript cDNA Synthesis Kit according to the manufacturer instructions (Biorad Laboratories). Newly primers were designed for choline dehydrogenase (CHDH), betaine aldehyde dehydrogenase (BADH), BHMT, dimethylglycine dehydrogenase (DMGDH), sarcosine dehydrogenase (SARDH), SLC6A12 (primers upon request). Quantitative (Q)-PCR was performed on a thermocycler CFX384 Biorad C1000 (annealing temperature 60°C and 40 cycles) with SYBRgreen as fluorophore. A melting curve between 65°C–95°C was performed to verify the quality of the amplification. Each sample was quantified in duplicate. RPL13 was used as reference gene.

We extracted total RNA from 5 to 10 mg of liver and brain tissues using the RNeasy Lipid Tissue Mini Protocol Kit from Qiagen according to the manufacturer instructions (Qiagen, Hilden, Germany). RNAs were quantified using a Nanodrop 2000 (Thermo scientific, Waltham, Maryland, USA). We performed RT-PCR using 1 µg of RNA with the iScript cDNA Synthesis Kit according to the manufacturer instructions (Biorad Laboratories, Hercules, California, USA). Newly primers were designed (supplemental data) for genes involved in the methionine metabolism (MTHFR, MAT1A, MAT2A, MAT2B, MTR, MTRR, SAHH, CBS), the phosphatidylcholine synthesis through the Kennedy pathway (CHKA, CHKB, CHPT1, PCYT1A, PCYT1B, PCYT2), the betaine metabolism (BADH, CHDH, BHMT, BHMT2, DMGDH, SDH), the folate metabolism (SHMT1, SHMT2, MTHFD1, MTHFD1L, MTHFD2), the vitamin B12 metabolism (MMACHC, MMADHC, LMBRD1) and for the genes of the main methyltransferases (GAMT, GNMT, PEMT) (Supplementary Table S1). Quantitative-PCR was performed on a thermocycler CFX384 Biorad C1000 (annealing temperature 60 °C and 40 cycles) with SYBRgreen as fluorophore. A melting curve between 65 and 95 °C was performed to verify the quality of the amplification. Each sample was quantified in duplicate. GAPDH and RPL13a were used as reference gene in liver and brain, respectively. Results are expressed in ∆∆Ct.