Iq5 rt qpcr detection system

Total RNA in the CSF and serum samples was extracted using TRIzol^® (Invitrogen Life Technologies, Carlsbad, CA, USA) with miRcute miRNA isolation kits (Tiangen Biotech, Beijing, China). The purity of the samples was measured at A260/A280 with an ultraviolet spectrophotometer (DR 6000™ UV VIS; HACH, Loveland, CO, USA), and cDNA was obtained by reverse transcription with an miRcute miRNA First-Strand cDNA Synthesis kit (Tiangen Biotech). The RT-qPCR was performed with an iQ5 RT-qPCR detection system (Bio-Rad Laboratories, Inc., Hercules, CA, USA). The reaction conditions for VEGF were as follows: Pre-denaturation at 94°C for 2 min; denaturation at 94°C for 30 sec, annealing at 55°C for 30 sec and extension at 71°C for 1 min, (35 cycles); and a final extension step at 71°C for 2 min. The RT-qPCR reaction conditions for miRNA-210 detection were as follows: Pre-denaturation at 95°C for 10 min; denaturation at 95°C for 15 sec, annealing at 60°C for 1 min and extension at 72°C for 2 min (40 cycles); and a final extension step at 72°C for 7 min. Results were calculated using the 2^−ΔΔCt method, and β-actin and U6 were used as the controls for VEGF and miRNA-210, respectively. The RT-qPCR primers (Tiangen Biotech) for the measurement of VEGF mRNA and miRNA-210 expression are listed in Table I.

RNA was collected from temporal cortex of AD mice by the use of TRIzol (Invitrogen, USA). The RNA concentration was measured under Nanodrop ND-1000 spectrophotometry (Nanodrop Tech, USA) and RNA integrity was detected with denatured agarose gel electrophoresis. cDNA was acquired by reverse transcription using the SuperScript VILO cDNA Kit. The primers were constructed and synthesized by Sangon Biotechnology (Shanghai, China). RT-qPCR was conducted with the iQ5 RT-qPCR Detection System (Bio-Rad Laboratories, USA) following manufacturer’s instructions. In this, all the primers were listed in Table 1.

RNA was collected from primary neuron cells by the use of TRIzol (Invitrogen, USA). The RNA concentration was the measured under Nanodrop ND-1000 spectrophotometry (Nanodrop Tech, USA) and RNA integrity was detected with denatured agarose gel electrophoresis. cDNA was acquired by reverse transcription using the SuperScript VILO cDNA Kit. The primers were constructed and synthesized by Sangon Biotechnology (Shanghai, China). RT-qPCR was conducted with the iQ5 RT-qPCR Detection System (Bio-Rad Laboratories, USA) following manufacturer’s instructions. Quantitative analysis of SLC7A11 and GPX4 was carried out using the SYBR Green Master Mix.