Nucleic acid protein analyzer

Cells were harvested and washed twice with ice-cold PBS, and the pellets were collected in 1X lysis buffer [50 mmol/l Tris-HCl (pH 6.8), 10% glycerol, 2% SDS, 0.25‰ bromophenol blue, and 0.1 mol/l DTT] was added for 100 µl/5×10⁶ cells. After heated at 95°C for 20 min, the lysates were centrifuged at 12,000 rpm for 10 min and the supernatant was collected. The protein concentration was determined by nucleic acid-protein analyzer (Beckman). Equal amount of lysate protein was separated on 8–12% SDS-PAGE and transferred onto polyvinylidene difluoride membrane (Pall). The non-specific binding sites were blocked with TBST buffer containing 5% non-fat dry milk for 2 h at room temperature. The membranes were incubated overnight at 4°C with specific primary antibodies, and the membranes were then washed thrice with TBST buffer and incubated at room temperature for 1 h with horseradish peroxidase-conjugated secondary antibody. After three washes with TBST buffer, the immuno-blots were visualized by the enhanced Phototope-Horseradish Peroxidase Detection kit purchased from Cell Signaling Technology and exposed to Kodak medical X-ray processor (Kodak, Rochester, NY, USA) (23 (link)).

The TRIzol one-step method (Invitrogen; Thermo Fisher Scientific, Inc.) was used to extract total RNA from KGN cells. Total RNA concentration was detected using a nucleic acid protein analyzer (Beckman Coulter, Inc.). RT was immediately performed using the Prime Script RT-PCR kit (Takara Biotechnology Co., Ltd.) according to the manufacturer's instructions to avoid RNA degradation. qPCR analysis was performed using the quantitative SYBR-Green PCR kit (Qiagen GmbH) and the Mx4000 quantitative PCR system (Stratagene; Agilent Technologies, Inc.). The reaction conditions used for the qPCR were as follows: Initial denaturation for 5 min at 95˚C; followed by 40 cycles of denaturation at 95˚C for 10 sec, annealing at 60˚C for 30 sec and extension at 72˚C for 34 sec. The internal controls used were GAPDH or U6. Gene expression was analyzed using the 2^-ΔΔCq method (17 (link)). The primer sequences for PCR were listed as follows: GAPDH forward, 5'-CTTTGGTATCGTGGAAGGACTC-3' and reverse, 5'-GTAGAGGCAGGGATGATGTTCT-3'; U6 forward, 5'-GCTTCGGCAGCACATATACTAAAAT-3' and reverse, 5'-CGCTTCACGAATTTGCGTGTCAT-3'; miR-451a forward, 5'-ACACTCCAGCTGGGAAACCGTTACCATTAC-3' and reverse, 5'-CTCAACTGGTGTCGTGGAGTCGGCAATTCAGTTGAGCTTACAG-3; ATF2 forward, 5'-TACAAGTGGTCGTCGG-3' and reverse, 5'-CGGTTACAGGGCAATC-3'; and cyclin D1 forward, 5'-CCGTCCATGCGGAAGATC-3 and reverse, 5'-GAAGACCTCCTCCTCGCACT-3'.

Total RNA was extracted from plasma of 56 IA patients and 56 controls using a QIAamp circulating nucleic acid kit (Qiagen, Hilden, Germany) according to the manufacturer's protocol. RNA quality and concentration were determined using Nucleic Acid/Protein Analyzer (DU730, Beckman Coulter, Inc). After reverse transcription of cDNA, real-time PCR was done in Mastercycler ep realplex (Eppendorf, Hamburg, Germany) using a QuantiFast SYBR Green PCR Kit (Qiagen, Hilden, Germany). All primers were synthesized by Ribobio Corp. (Guangzhou, China). U6 was used to normalize the let-7 level in both cases and controls. Each sample was analyzed in triplicate. The relative expression of let-7 family was described using the 2^–ΔCt method.^{25 (link)}

Total RNA was extracted from cells and ipsilateral cortex samples with TRIzol (Invitrogen; Thermo Fisher Scientific, Inc.). The concentration of total RNA was detected using a nucleic acid protein analyzer (Beckman Coulter, Inc.). To avoid RNA degradation, reverse transcription was performed immediately with the Prime Script RT-PCR kit (Takara Bio, Inc.) according to the manufacturer's instructions. qPCR was performed on the Eppendorf Real Plex 4 instrument (Eppendorf) using real-time SYBR Green (cat. no. 1708882AP; Bio-Rad Laboratories, Inc.). The specific primers designed by Invitrogen (Thermo Fisher Scientific, Inc.) with the following sequences: NQO1 forward, 5′-CATTCTGAAAGGCTGGTTTGA-3′ and reverse, 5′-CTAGCTTTGATCTGGTTGTCAG-3′; HO-1 forward, 5′-ATCGTGCTCGCATGAACACT-3′ and reverse, 5′-CCAACACTGCATTTACATGGC-3′; TNF-α forward, 5′-TGATCCGCGACGTGGAA-3′ and reverse, 5′-ACCGCCTGGAGTTCTGGAA-3′; IL-6 forward, 5′-CCAAGAGGTGAGTGCTTCCC-3′ and reverse, 5′-CTGTTGTTCAGACTCTCTCCCT-3′ and β-actin forward, 5′-CCGTGAAAAGATGACCCAGA-3′ and reverse, 5′-TACGACCAGAGGCATACAG-3′. The thermocycling conditions were as follows: 95°C for 10 min followed by 40 cycles of 95°C for 15 sec then 60°C for 1 min. mRNA levels were quantified using the 2^−ΔΔCq method (25 (link)).