Iq5 optical system

Genomic DNA was isolated from brain regions ± 1.0 mm from the LPS injection site (average tissues weight ranged from 50–60 mg) using QIAamp DNA mini kit (Qiagen) following the manufacturer’s instructions. The amount of donor-derived cell DNA was quantitatively determined by real-time qPCR with primers and probe specific to male murine Y chromosome [27 (link)]. Real-time qPCR was performed on an iQ5 optical system (BioRad) using forward primer 5’-TTTTGCCTCCCATAGTAGTATTTCCT-3’, reverse primer 3’-TGTACCGCTCTGCCAACCA-3’ and the TaqMan probe 5’-/56-FAM/AGGGATGCC/ZEN/CACCTCGCCAGA-/3IABkFQ/-3’ (Integrated DNA Technologies). Standard curves were generated by serially diluting DNA from male mouse brain. DNA isolated from female mouse brain was included as a negative control in every assay. The average DNA content in a diploid mouse cell range from 5 to 7 pg [28 (link)], therefore a parameter of 6 pg gDNA per cell was applied to convert DNA to cell numbers presented in all results.

The involved reference genes were downloaded from the NCBI (http://www.ncbi.nlm.nih.gov/) and transcriptome data of C. megacephala. The candidate primers were designed by online primer design tool for Real-time PCR (http://www.idtdna.com/site) with default settings. The Primers were synthesized by Newtsingke Biotech (Wuhan, China). The length and identity of PCR products were assessed with gel electrophoresis and sequence analysis. Then sensitivity, specificity, and capacity of the right primers were testified by melting curve and standard curve with the Bio-Rad iQ5 Optical System. Then the remaining qualified primers were used for further qPCR evaluation. A standard curve was achieved for each gene by five-fold serial dilution of the templates. The qualified primers used for qPCR, their PCR efficiency and regression coefficient and were shown in Table 1.

Genomic DNA was extracted for genotyping using PCR, as previously reported (Edwards et al., 1991 ). Total RNA was prepared with TRIZOL reagent (Invitrogen, Carlsbad, CA, USA) and TURBO DNA-free Kit (Applied Biosystems, Foster City, CA, USA). cDNA synthesis was performed with Ready-To-Go Kit (GE Healthcare Life Sciences, Stockholm, Sweden) and analyzed by reverse transcription PCR (RT-PCR). PCR primers were designed by Primer3². Quantitative real-time PCRs (qPCRs) with iQ SYBR Green Supermix (Bio-Rad, Techview, Singapore) were performed by using MyiQ thermocycler and iQ5 optical system (Bio-Rad). qPCR data were normalized to the internal control of PP2AA3 (PP2A) (Czechowski et al., 2005 (link)).

After either 6 or 12 h of exposure to the various experimental conditions, VSMC were washed with cold PBS, scraped, and collected. RNA was isolated from samples via RNAeasy Protect Mini Kit per manufacturer’s instructions (Qiagen, Germantown, MD). RNA concentrations were determined via a Gen5 Take 3 Module per manufacturer’s instructions (BioTek, Winooski, VT). Equivalent RNA amounts were converted into cDNA via a QuantiTect Reverse Transcription Kit per manufacturer’s instructions (Qiagen). Quantitative PCR was performed via a QuantiTect SYBR Green PCR Kit using SOD-1 and GAPDH primers according to manufacturer’s instructions (Qiagen). Fluorescence was detected via an iQ5 optical system (Bio-Rad) and Ct values were exported as Excel files. Expression of sod-1 was calculated using GAPDH as a reference and normalized to male control.

miRcute miRNA isolation, miRcute miRNA first-strand cDNA synthesis and miRcute miRNA qPCR detection kits were purchased from Tiangen Biotech Co., Ltd. (Beijing, China). A quantitative polymerase chain reaction (qPCR) iQ5 Optical System and Image Lab^™ software were obtained from Bio-Rad Laboratories, Inc. (Hercules, CA, USA). The primary antibodies against HIF-1α were purchased from Abcam (Cambridge, MA, USA).

Isolated tumor cells from tumor tissue or cultured cells were lysed in TRIzol reagent (Life Technologies). Total RNAs were extracted using the method recommended by the manufacturer. Reverse transcription polymerase chain reaction was performed using the Superscript first-strand synthesis system (Life Technologies). Quantitative real-time PCR was set up with 0.5 μl of cDNA (from a total of 25 μl of each reverse transcription reaction) and primers at a concentration of 10 nm; the reactions were carried out for 40 cycles using SYBR Green and measured using an iQ5 Optical System (Bio-Rad, Hercules, CA, USA).
The primers for quantitative real-time PCR have been synthesized by Sangon Biotech (Shanghai, China). The primer sequences for mouse IL-6 are 5′-CAGAAGGAGTGGCTAAGGACCA-3′ and 5′-ACGCACTAGGTTTGCCGAGTAG-3′. The primer sequences for mouse VEGF-A are 5′-GGAGATCCTTCGAGGAGCACTT-3′ and 5′-GGCGATTTAGCAGCAGATATAAGAA-3′. The primer sequences for mouse PDGF-β are 5′-TCTCTGCTGCTACCTGCGTCTG-3′ and 5′-GGAAGTTGGCGTTGGTGCGATC-3′, and the primer sequences for mouse β-actin are 5′-TGTGCTGTCCCTGTATGCCTCT-3′ and 5′-GGAA CCGCTCGTTGCCAATAGT-3′. Quantitative real-time PCR reactions were performed in triplicate, and β-actin was used as an internal control. The cycle threshold values were converted to relative gene expression levels via the 2^−ΔΔCt method.