Taqman method

We extracted DNA from the buffy coat fraction with a DNA Blood mini kit (Qiagen, Tokyo, Japan) or BioRobot EZ1 and EZ1 DNA Blood 350 mL Kit (Qiagen). We genotyped ALDH2 Glu504Lys polymorphism (rs671) by the TaqMan method (Applied Biosystems, Foster City, CA, USA). The quality control of genotyping was assessed statistically by using the Hardy-Weinberg test and by retyping of a random sampling of 5% of subjects.

Quantitative real time PCR was carried out in triplicate using a Model 7500 fast real time PCR system and the TaqMan method (Applied Biosystems, Foster, CA). Primers and probes for human BCO1 (Hs00363176_ml), 18S rRNA (Hs99999901_sl) and GAPDH (Hs02758991_g1) were obtained from Applied Biosystems. Relative fold changes of gene expression compared with the internal controls 18s rRNA and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were determined using 2^-ΔΔCt method as described by Livak K.J. et al [35 (link)]. Briefly, the fold change was calculated as follow: relative fold changes = 2 –^ΔΔCt, where ΔΔCt = (ΔCt _treated)– (ΔCt _untreated), ΔCt = Ct _target−Ct _18srRNA or Ct _GAPDH.

Genotyping was carried out with genomic DNA isolated from human leukocytes using a commercial kit (QIAamp DNA Blood Mini Kit QIAGEN, Hilden, Germany), as previously reported^{13 (link)}. A genetic variant was analyzed using genomic DNA for a C/T variant of C9orf3 (rs4385527) with the StepOnePlus real-time PCR system and the TaqMan method (Applied Biosystems, Foster City CA, USA). The probe mix (primer) of rs4385527 was TaqMan SNP Genotyping Assay (Assay ID: C___7961143_20). The global minor allele frequency of C9orf3 (rs4385527) was 0.23 (T allele). The Japanese minor allele frequency of C9orf3 (rs4385527) was 0.21 (T allele). The real-time PCR was analyzed by using StepOnePlus software version 2.2.2. The PCR program consisted of Pre-PCR Read (Holding Stage 1) at 50 °C for 2 min, Holding Stage 2 at 95 °C for 10 min, 45 cycles of Cycling Stage (denaturing at 92 °C for 15 s followed by annealing and extension at 60 °C for 1 min), and post-PCR Read (Holding Stage 3) at 60 °C for 30 s.

RNA extraction, cDNA preparation, and real time PCR analysis were performed as in the work of [40 (link)] on QuantStudio 5 Real-Time PCR System (Applied Biosystems, Foster City, CA, USA) using SYBR Green quantification. The relative amount of each gene was measured as fold change using the 2^−ΔΔCt method (β-Actin or P0 served as endogenous control). Expression of β-Actin, GLUT1, and primiR-675 were evaluated by TaqMan method (Applied Biosystems). Primers for MALAT1 was as in the work of [40 (link)]. Primers sequences were as follows: 91H 5′-GGCCCTGAATCAAACATCATG-3′ and 5′-TCACGCAGCGTTGCTCTCT-3′, CDH1 5′-CTGGGACTCCACCTACAGAAAGTT-3′ and 5′-CCAGAAACGGAGGCCTGAT-3′, EPO 5′-AGCCCAGAAGGAAGCCATCT-3′ and 5′-GAAAGTGTCAGTGATTGTTC-3′, H19 5′-TCAAGCCTGGGCCTTTGAAT-3′ and 5′-GGCTGATGAGGTCTGGTTCC-3′, ITGA2 and 5′-CAGCAATGTGGGAATCAGTATTACA-3′ and 5′-GGAAGGGCAGGGCTGAGT-3′, ITGB3 5′-GAAAACCCCTGCTATGATATGAAGAC-3′ and 5′-GTTAGCGTCAGCACGTGTTTG-3′, ITGB4 5′-TGAAGAGCTGCACGGAGTGT-3′ and 5′-GGTCCCTGAACATCTCGTCTGT-3′, KDR 5′-CCAGCAAAAGCAGGGAGTCT-3′ and 5′-TGGTAGCCGCTTGTCTGGTT-3′, P0 5′-TCGACAATGGCAGCATCTAC-3′ and 5′-ATCCGTCTCCACAGACAAGG-3′, RUNX2 5′-CCCGTGGCCTTCAAGGT-3′ and 5′-TGACAGTAACCACAGTCCCATCTG-3′, HIF1α 5′-GAACAAAACACACAGCGAAGCT-3′ and 5′-TGCAGTGCAATACCTTCCATGT-3′, HIF2α 5′- ATCAGCTTCCTGCGAACACA-3′ and 5′-CTTCGGCTTCGGACTCGTT-3′, CCND1 5′-CCGTCCATGCGGAAGATC-3′ and 5′-TGCAGGCGGCTCTTTTTC-3′.