The total RNA was extracted from tissues using RNAEX reagent (Accurate Biotechnology, China). It was reverse transcribed into cDNA by using Evo M-MLV RT premix (Accurate Biotechnology, China) and quantitative real-time polymerase chain reaction (RT-PCR) system (TaKaRa, Japan). Then a SYBR Green Premix Pro Taq HS qPCR Kit (Accurate Biotechnology, China) was used for PCR. The PCR liquid volume was 20 μL. Quantitative PCR was performed using an RT-PCR system (BioRad, Singapore). At 95℃, 40 cycles were amplified after 90 s of initial denaturation, 10 s desaturation at 95℃, and 34 s of extension at 60℃. GAPDH was used as the reference gene for calculation. And the primer sequences are shown in Table 1 .
Quantitative real time polymerase chain reaction
Manufactured by Takara Bio
Sourced in China
Quantitative real-time polymerase chain reaction (qRT-PCR) is a laboratory technique used to detect and quantify the amount of a specific DNA or RNA sequence in a sample. It combines the amplification of a target sequence with the simultaneous measurement of the amplified product.
Automatically generated - may contain errors
Lab products found in correlation
Rt pcr system, by Takara Bio (1 mentions)
Rnaex reagent, by Accurate Biology (1 mentions)
Rt pcr system, by Bio-Rad (1 mentions)
Sybr green premix pro taq hs qpcr kit, by Accurate Biology (1 mentions)
Evo m mlv rt premix, by Accurate Biology (1 mentions)
Primescript rt master mix, by Takara Bio (1 mentions)
Trizol reagent, by Vazyme (1 mentions)
2 protocols using quantitative real time polymerase chain reaction
1
RNA Extraction and RT-qPCR Quantification
2
Quantitative Analysis of Spinal Cord Gene Expression
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Under deep anesthesia, the L4-L5 spinal cord segments of the mice were quickly removed and analyzed. Total RNA was isolated with Trizol reagent (R401-01; Vazyme, Nanjing, China) according to the manufacturer's instructions. cDNA was then synthesized using 5X PrimeScript RT Master Mix (TaKaRa, Dalian, China). Quantitative real-time polymerase chain reaction (TaKaRa) was performed in a 20-μL reaction mixture consisting of 10 μL of 2× SYBR Premis Ex TaqTM II, 2 μL of cDNA, 1 μL of forward primer, 1 μL of reverse primer and 6 μL of ddH2O. The thermal cycling conditions used were 95°C for 5 min, 40 cycles of 95°C for 5 s and 58°C for 30 s, and 72°C for 30 s. The specific primers used for detection are listed in Supplementary Table S1. Relative mRNA levels were calculated using the 2 -ΔΔCT method [30] . Gene expression was normalized to that of the housekeeping control gene GAPDH.
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