Moloney murine leukaemia virus reverse transcriptase

Total RNA was extracted from the hippocampus using the NucleoSpin RNA Kit (Takara, Otsu, Japan) according to the manufacturer’s instructions, and was then reverse transcribed into cDNA using Oligo-T priming and Moloney murine leukaemia virus reverse transcriptase (Takara). Quantitative polymerase chain reaction was performed using the FastStart Essential DNA Probes Master Mix (Roche, Basel, Switzerland) and run on a Step One Plus Real Time Polymerase Chain Reaction System using the Taqman gene expression assay mix (Applied Biosystems, Carlsbad, CA). Target gene mRNA expression was normalised to β-actin mRNA expression, and the relative amounts of all mRNAs were calculated using the comparative Ct method. The primer sequences and reaction parameters are shown in Table 1.

Total RNA was extracted from human cardiovascular tissue samples using the miRNeasy Mini Kit (Qiagen, Beijing, China) and was converted to cDNA using random hexamers, oligo(dT) primers and Moloney murine leukaemia virus reverse transcriptase (TaKaRa, Dalian, China). WNT5A mRNA levels were measured with qPCR using the StepOnePlus system (ABI, Foster City, CA), with GAPDH as an internal reference gene. The reaction mixture contained 10 μM of each primer, 2 × SYBR Green PCR Master Mix (ABI, CA, USA) and 1 μl cDNA. The primers that were used are listed in Supplemental Table 2. Each reaction was performed in triplicate.

The sequence of human LRP2 used in the siRNA studies were: sense 5′GCAGCUUACUUGUGACAAU3′ and antisense 5′AUUGUCACAAGUAA GCUGC3′, which were synthesized and purified by RiboBio Co., Ltd. Scrambled siRNA (Cat. no: siN05815122147, RiboBio, Guangzhou, China) was used as a control for comparison. 6 × 10⁵ HEK293T cells were seeded in 6-well plates and transfected with 100 nM siRNA and 400 ng 77Lys/77Met TCN2 plasmid. After 48 h, total RNA was extracted from the transfected cells using the miRNeasy Mini Kit (Qiagen, Beijing, China) and was converted to cDNA using random hexamers, oligo (dT) primers and Moloney murine leukaemia virus reverse transcriptase (TaKaRa, Dalian, China). LRP2 mRNA levels were measured using qPCR with the StepOnePlus system (ABI, Foster City, CA). These studies utilized GAPDH as an internal reference gene. The reaction mixture contained 10μM of each primer, 2× SYBR Green PCR Master Mix (ABI, CA, USA) and 1 μl cDNA. The primers used are listed in Supplementary Table 2. Each reaction was performed in triplicate.