Total RNA was isolated from cells using a TRIZOL Regent Kit (Takara, Tokyo, Japan) according to the manufacturer’s instructions. Approximately 1 µg of total RNA was used for cDNA syntheses using a standard reverse transcription kit (Takara, Tokyo, Japan). The inverse transcription process was as follows: 25 °C for 10 min, 42 °C for 15 min, and 85 °C for 5 min. The primers used (designed by AlleleID 6) are shown in Table 1 (synthesized in Comate Bioscience Co. Ltd., Changchun, China). qPCR was carried out in a 7500c real-time PCR detection system (Applied Biosystems, Carlsbad, CA, USA) with the SYBR premix EX Taq (TaKaRa). GAPDH was regarded as the control.
Trizol regent kit
Manufactured by Takara Bio
Sourced in Japan
TRIZOL Regent Kit is a reagent designed for the isolation and purification of total RNA from a variety of biological samples, including cells, tissues, and microorganisms. It is a mono-phasic solution of phenol, guanidine isothiocyanate, and other proprietary components that facilitate the extraction and separation of RNA from DNA, proteins, and other cellular components.
Automatically generated - may contain errors
Lab products found in correlation
Standard reverse transcription kit, by Takara Bio (2 mentions)
Sybr premix ex taq, by Takara Bio (2 mentions)
7500c real time pcr detection system, by Thermo Fisher Scientific (1 mentions)
Nanodrop 2000, by Thermo Fisher Scientific (1 mentions)
Sybr premix ex taq 2, by Takara Bio (1 mentions)
Primescript rt reagent kit with gdna eraser, by Takara Bio (1 mentions)
7500 fast real time pcr system, by Thermo Fisher Scientific (1 mentions)
4 protocols using trizol regent kit
1
RNA Isolation and qPCR Analysis
2
Gene Expression Analysis by qRT-PCR
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Total RNA was extracted from cells using a TRIZOL Regent Kit (Takara, Tokyo, Japan) according to the manufacturer’s instructions. And RNA concentration was detected by Nanodrop 2000 (Thermo, Massachusetts, United States). Then RNA was reverse transcribed to cDNA using a standard reverse transcription kit (Takara, Tokyo, Japan) according to the instructions. The primers were designed by the NCBI primer design tool, and are shown in Table 1 (synthesized in Comate Bioscience Co. Ltd., Changchun, China). qPCR was performed using the SYBR premix EX Taq (TaKaRa). Relative gene expression was normalized by GAPDH using the 2−ΔΔCt method.
3
RNA Extraction and RT-qPCR Analysis of Ischemic Hippocampus
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Total RNA of hippocampus of ischemic brain were extracted using a Trizol regent kit (TaKaRa Bio Inc., Japan) and cDNA was prepared via reverse-transcription using the PrimeScript™ RT Reagent Kit with gDNA Eraser (TaKaRa Bio Inc.), according to manufacturer's protocol. Real-time quantitative polymerase chain reaction (RT-qPCR) was performed in a 10 μL volume using SYBR Premix Ex Taq™ II (TaKaRa Bio Inc.). The following cycling conditions were used: 30 s at 95°C followed by 40 cycles of 5 s at 95°C and 30 s at 60°C. The specific primers are listed as Table 1 . Gene expression was determined relative to the housekeeping gene GAPDH using the 2−ΔΔCt method.
4
NLRP3 Expression Quantification by RT-qPCR
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The effect of pNLRP3 transfection was determined by RT-qPCR. Total RNA was extracted using the TRIzol regent kit (TaKaRa Bio, Japan), and cDNA was prepared by reverse transcription. PCR was performed on a 7500 FAST Real-Time PCR System (Applied Biosystems, Foster City, CA, USA) with specific primers and the RT-qPCR Assay Kit (TaKaRa Bio, Japan). Primer sequences of the targeted genes used in this study were as follows: NLRP3 (5′-ATTACCCACCCGAGAAAGG-3′, forward; 5′-CATGAGTGTGGCTAGATCCAAG-3′, reverse) and β-actin (5′-CACCCGCGAGTACAACCTTC-3′, forward; 5′-CCCATACCCACCATCACACC-3′, reverse). NLRP3 expression levels were normalized for expression of β-actin and expressed as the fold ratio compared with the control group.
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