Total RNA was isolated with TRIzol reagent (Invitrogen) according to the manufacturer’s protocol. A PrimeScript RT reagent kit (TaKaRa, Japan) was used to synthesize cDNA from total RNA, and then, qRT-PCR was performed with a miScript SYBR Green qRT-PCR kit (QIAGEN, Germany) on an ABI 7900 fast real-time PCR system (Applied Biosystems, Carlsbad, USA). Expression levels were normalized to that of GAPDH, and the 2−ΔΔCT method was used to analyze the relative gene expression. The specific primers were designed by RiboBio (Guangzhou, China) with the following sequences: TNF-α (forward: 5′-TCCCAGGTTCTCTTCAAGGGA-3′, reverse: 5′-GGTGAGGAGCACGTAGTCGG-3′), IL-1β (forward: 5′-GCCTCGTGCTGTCGGACCCATAT-3′, reverse: 5′-TCCTTTGAGGCCCAAGGCCACA-3′), IL-6 (forward: 5′-AAAGAGTTGTGCAATGGCAATTCT-3′, reverse: 5′-AAGTGCATCATCGTTGTTCATACA-3′), CCL2 (forward: 5′-CTTCTGGGCCTGCTGTTCA-3′, reverse: 5′-CCAGCCTACTCATTGGGATCA-3′) and CCR2 (forward: 5′-AGAGGCGAAGGCAACAGTCG-3′, reverse: 5′-GCAGGGCCAATGTCTAGTCC-3′).
Miscript sybr green qrt pcr kit
Manufactured by Qiagen
Sourced in Germany
The MiScript SYBR Green qRT-PCR kit is a reagent kit designed for quantitative real-time reverse transcription PCR (qRT-PCR) analysis. It contains the necessary components for the detection and quantification of RNA targets, including a SYBR Green-based master mix and a reverse transcriptase enzyme.
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Lab products found in correlation
2 protocols using miscript sybr green qrt pcr kit
1
qRT-PCR Analysis of Inflammatory Markers
2
Isolation and Quantification of miRNA
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Total RNA was isolated from cultured cells and tissues using the TRIzol reagent (Life Technologies, Grand Island, NY, USA). Total RNA was isolated from the culture medium (CM) of cultured cells and from serum using the TRIzol LS (Life Technologies) reagent according to the manufacturer's protocol. Reverse transcription of miRNA was performed by using the Reverse Transcript Kit (QIAGEN, Duesseldorf, Germany). For gene expression analysis, qRT-PCR was performed using the miScript SYBR Green qRT-PCR kit (QIAGEN). β-actin (ACTB) and U6 were used as an endogenous control for cultured cells and tissue samples. Fold changes were calculated using the 2ΔΔCt method. The compilation of data was transformed using base 2 logarithmic transformation. After the transformation, the data closely followed normal distribution. For CM and serum samples, a standard curve was made for the endogenous control and quantification. All reactions were performed in triplicate. All of the oligonucleotides and primers used in the experiments are listed in Tables S1 and S2.
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