Sds plate utility v2

Genomic DNA was isolated from whole blood cells using Puregene^® DNA Purification Kit (Cat. #D550, Gentra, USA). Firstly, 5-15 μl of gDNA was extracted from 300 μl of whole blood sample. The ACE polymorphism (rs1799752) analysis was performed using the MGB TaqMan^® SNP Genotyping assay with 3 types of primers and 2 types of probes^{32 (link)}. The nucleotide sequences utilized in the study are as follows: Primer ACE111: 5’-CCC-ATC-CTT-TCT-CCC-ATT-TCT-C-3’, Primer ACE112: 5’-AGC-TGG-AAT-AAA-ATT-GGC-GAA-AC-3’, Primer ACE113: 5’-CCT-CCC-AAA-GTG-CTG-GGA-TTA-3’, the I-allele-specific sequence: VIC-5’AGG-CGT-GAT-ACA-GTC-A-3’-MGB, and the D-allele-specific sequence: FAM-5’TGC-TGC-CTA-TAC-AGT-CA-3’MGB. The reaction components necessary for the amplification mix were 10-50 ng gDNA, TaqMan^® Genotyping Master Mix, 10 pmol/each primer, 150 nM VIC probe, and 75 nM FAM probe. Finally, target gene amplification was performed using the ABI 7900HT Real-Time PCR system (Applied Biosystems^®, USA). The cycling profile included the initial denaturation at 92 ℃ for 15 seconds, followed by 40 cycles of annealing and extension at 57 ℃ for 1 minute. The calculation for the genotypes was performed automatically (SDS Plate Utility v2.2 software, Applied Biosystems^®, USA).

Gene expression validations of interest were performed on RNA from biological replicates, extracted using Qiagen kits, and reverse transcribed using SuperScript III Reverse Transcriptase (Invitrogen, Waltham, MA, USA). Primers for RT2 qPCR were purchased from Qiagen (Valencia, CA, USA), for SIRT1 (catalog number PPH02188A-200) and APP (catalog number PPH05947A-200). PCRs were performed using RT² SYBR Green ROX FAST Mastermix (Qiagen) in a 7900HT Fast Real-Time PCR System with Fast 96-Well Block Module (Applied Biosystems, Foster City, CA, USA) with an SDS Plate utility v2.2 software (Applied Biosystems). The results were normalized to the geometric mean of both glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and 18S ribosomal RNA housekeeping genes, with PrimeTime qPCR assay oligomers (catalog numbers Hs.PT.39a.22214836, and Hs.PT.39a.22214856, respectively, Integrated DNA Technologies, San Diego, CA, USA).

Total RNA was isolated from the cells using RNAeasy Mini kit (Qiagen), according to the manufacturer’s instructions. Total RNA concentration was measured using the Nanodrop spectrophotometer and then used for reverse transcription using SuperScript III Reverse Transcriptase (Invitrogen, Waltham, MA). Most primers were purchased from Qiagen (Valencia, CA). PCRs were performed using RT2 SYBR Green ROX FAST Mastermix (Qiagen), in a 7900HT Fast Real-Time PCR System with Fast 96-Well Block Module (Applied Biosystems, Foster City, CA), with a SDS Plate utility v2.2 software (Applied Biosystems). The results were normalized to the expression of GAPDH. The primers for the DA receptors were previously described [18 (link)]. Primers for CCR5 were purchased from Qiagen.