Rotor gene q real time pcr system

A total of 1–2 μg RNA was treated with RNase-Free DNase Set (QIAGEN) to remove contaminating DNA and then subjected to cDNA synthesis using the SuperScript II RT Kit (Life Technologies) according to the manufacturer’s instructions. qRT-PCR was performed on a Rotor-Gene Q real-time PCR system (QIAGEN). QuantiFast SYBR Green PCR master mix (QIAGEN) was used for amplification. Zoysiagrass β-ACTIN (GU290545.1) sequence was used as an internal control to measure the relative amount of transcripts. Information on oligonucleotide sequences for qRT-PCR analysis is listed in S10 Table.

Total RNA was isolated from 20 mg of hippocampal CA1 tissue using RNeasy Mini Kit (Qiagen, Germantown, MD) according to the manufacturer’s instructions. The RNA samples were resuspended in nuclease-free water and quantified spectrophotometrically at 260 nm. All RNA samples had an A260:A280 value between 1.8 and 2.0. cDNA synthesis was carried out by using the QuantiTect Reverse Transcription Kit (Qiagen) according to the manufacturer's protocols. The cDNA stock was stored at -20 °C. Quantitative PCR for TPM2 and the endogenous control gene HPRT was carried out using the iQ SYBR Green Supermix (Bio-rad). The primer sequences for HPRT were: 5'-GCCGACCGGTTCTGTCAT-3' (forward) and 5'-TCATAACCTGGTTCATCATCACTAATC-3' (reverse). The primer sequences for TPM2 were: 5'-AAGGGGACAGAGGATGAG-3' (forward) and 5'-CTTTCTCAGCCTCCTCCA-3' (reverse). Amplification was performed using the Rotor-Gene Q Real Time PCR system (Qiagen), and the reaction condition followed the manufacturer’s protocols. The thermal cycler protocol used is as follows: 95 °C for 10 min, 95 °C for 10 s, and 60 °C for 30 s for 40 cycles. The cycle threshold (Ct) values and related data were analyzed using the Rotor-Gene Q Real Time PCR system Software (Qiagen). The expression level of TPM2 was normalized with that of HPRT. The relative expression levels (in fold) were determined using the 2-^(△△Ct) method.

Selection of SNP in HIF1A and WISP1 genes was conducted based on information from the dbSNP database (http://www.ncbi.nlm.nih.gov/projects/SNP/). Three polymorphisms were included for the HIF1A gene and one for the WISP1 gene in the genotyping tests (Table 1). Real-time PCR was performed in a total volume of 10 μL, that contained 40 ng DNA (1 μL), 1X TaqMan Universal PCR Master Mix (5 μL), TaqMan Probes 20X (0.5 μL), and water (3.5 μL). Thermal cycling conditions were as follows: 95 °C for 10 min, followed by 40 cycles at 92 °C for 15 s and at 60 °C for 1 min. The Rotor-Gene Q Real-time PCR System (QIAGEN, Hilden, Germany) was employed for data acquisition. Genotyping of the HIF1A and WISP1 polymorphisms were carried out using the 5′ exonuclease TaqMan Allelic Discrimination assay, which was performed utilizing minor groove binder probes fluorescently labeled with VIC or FAM and the protocol recommended by the supplier (Applied Biosystems, Foster City, CA, USA). Analysis for interpretation was performed with Rotor-Gene Q Series ver. 2.0.2 software.Table 1

Single-nucleotide polymorphism (SNP) studied

Gene	Chromosome position	dbSNP rs ID	Location	SNP type
HIF1A	Chr14: 62207557	rs11549465	Exon 12	Pro582Ser, C1772T
	Chr14: 62207575	rs11549467	Exon 12	Ala588Thr, G1790A
	Chr14: 62213848	rs2057482	3′ UTR	C191T
WISP1	Chr8: 134229883	rs2929970	3′ UTR	A2364G

Total RNA from tissue or cells was isolated with QIAGEN RNeasy®mini kit and the manufacturer's protocol (Qiagen, Germany). One μg of the RNA was reverse-transcribed to cDNA by M-MLV reverse transcriptase (Promega, WI, USA), to which SYBR Green PCR Master Mix (Enzynomics, Daejeon, Republic of Korea) was added. The primers for each gene are as follows: the primers for TNF-α were 5′-GGTCTGGGCCATAGAACTGA-3′ and 5′-CAGCCTCTTCTCATTCCTGC-3′; IL-1β primers were 5′-AGGTCAAAGGTTTGGAAGCA-3′ and 5′-TGAAGCAGCTATGGCAACTG-3′; IL-6 primers were 5′-TGGTACTC CAGAAGACCAGAGG-3′ and 5′-AACGATGATGCACTTGCAGA-3′; NQO-1 primers were 5′-GCAGTGCTTTCCATCACCC-3′ and 5′-TGGAGTGTGCCCAATGCTAT-3′; HO-1 primers were 5′-TGAAGGAGGCCACCAAGGAGG-3′ and 5′-AGAGGTCACC CAGGTAGCGGG-3′; GCLC primers were 5′-CACTGCCAGAACACAGACCC-3′ and 5′-AGGTCTGCTGAGAAGCCT-3′; and GAPDH primers were 5′-GGAGCCAAAAGG GTCATCAT-3′ and 5′-GTGATGGCATGGACTGTGGT-3′. The thermal reaction was run at 95°C for 10 min, followed by 40 cycles of 95°C for 10 s, 57°C for 15 s, and 72°C for 20 s in a Rotor-Gene Q real-time PCR system (Qiagen). The threshold cycles (Ct) were used to quantify the mRNA expression of the target genes.

Mice were deeply anesthetized with sodium thiopental 24 h after the induction of ischemia, after which they were perfused transcardially with cold PBS and the brain cortexes were collected. Total RNA was isolated from the ischemic cortex using TRIzol reagent^TM (Invitrogen, Carlsbad, CA) according to the manufacturer’s recommendations. The RNA was then reverse-transcribed for 1 h at 42 °C with Moloney Murine Leukemia Virus reverse transcriptase (Promega, Madison, WI) to produce cDNA. Real-time PCR was performed to quantify the amount of NOX2 and NOX4 mRNA in the ischemic brain using a Rotor-Gene Q real-time PCR system (Qiagen, Hilden, Germany) with SYBR Green PCR Master Mix (Qiagen), after which the results were normalized to GAPDH gene expression. All experiments were performed in triplicate and repeated at least three times. The following primer sequences were used:

mouse Nox2 -557 to-725 (175 bp), 5′-CTGAAGGGGGCCTGTATGTG-3′ (forward)-, and 5′-ATGGCAAGGCCGATGAAGAA-3′ (reverse);

mouse Nox4 -2478 to −5896 (119 bp), 5′-CCTCGCTGCAGTGTTCCTAA-3′ (forward)-, and 5′-GATTGGCTAAGGGGGAGCAG-3′ (reverse).

The threshold cycles (Ct) were used to quantify the mRNA expression of target genes.