Total RNA was extracted from colon tissues following the instructions of TRIzol reagent (Invitrogen). After the determination of RNA concentration by ND2000 (Thermo Fisher), 1 μg of RNA samples was used to generate cDNA using the TaKaRa RT reagent kit from Takara. Real‐time PCR was performed on Light Cycler 96 (Roche) with SYBR Green kit (Takara, Tokyo, Japan). Relative mRNA expression was calculated by 2−ΔΔCt, after normalization to the levels of U6 mRNA.
Takara rt reagent kit
Manufactured by Takara Bio
Sourced in United States, China, Japan
The TaKaRa RT reagent kit is a set of reagents used for the reverse transcription of RNA into complementary DNA (cDNA). The kit includes all the necessary components for the reverse transcription reaction, including a reverse transcriptase enzyme, reaction buffer, and primers.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (3 mentions)
Sybr green kit, by Takara Bio (2 mentions)
Nd 2000, by Thermo Fisher Scientific (1 mentions)
Lightcycler 96, by Roche (1 mentions)
Sybr green master mix, by Thermo Fisher Scientific (1 mentions)
Faststart essential dna green master, by Roche (1 mentions)
Mir x mirna first strand synthesis kit, by Takara Bio (1 mentions)
Hp total rna isolation kit, by Omega Bio-Tek (1 mentions)
Lightcycler 480 real time pcr detection system, by Roche (1 mentions)
Nanodrop nd 2000 spectrophotometer, by Thermo Fisher Scientific (1 mentions)
Trizol reagent, by Takara Bio (1 mentions)
5 protocols using takara rt reagent kit
1
Total RNA Extraction and qRT-PCR Analysis
2
Evaluating Neuroinflammation and Oxidative Stress
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The effect of the PML@B/Gel on inflammation, scavenged ROS, and pain was analyzed. The gene expression levels were assessed using qRT-PCR from neuroblastoma and nerve tissue. The cells under the same experimental conditions as the biocompatibility test were treated with TNF-α (50 ng/ml) for 24 h before retrieving mRNA. For mRNA isolation from nerve tissue, Trizol reagent (Invitrogen, USA) was used. Then the cDNA was synthesized by random hexamer primers and a TAKARA RT reagent kit (Takara, USA). SYBR Green Master Mix (Applied Biosystems, USA). The expression of target genes was normalized by 18S rRNA as a reference gene.
3
Quantitative RNA Expression Analysis
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Total RNA was isolated using the HP total RNA isolation kit (Omega bio-tek) following manufacturer’s instructions. 1 μg of total RNA was reverse-transcribed into cDNA using Takara®RT reagent Kit (Takara), and 0.8 μg of total RNA was used for miRNA reverse transcription with the Mir-X miRNA First-Strand Synthesis Kit (Takara). Quantitative PCR was performed using Roche FastStart Essential DNA Green Master (Roche) with the primers listed in Supplemental Table S8 . The relative gene expression was calculated using the equation 2-Δ(ΔCT) where ΔCt = Ct (mRNA)−Ct (actin) or Ct (miRNA)−Ct (U6).
4
Quantitative Gene Expression Analysis in Spleen Samples
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Total RNA was extracted from spleen samples using TRIzol reagent (TaKaRa, Dalian, China). The quality of total RNA was measured by using NanoDrop ND-2000 spectrophotometer (Thermo Scientific, Wilmington, DE, USA). For each sample, 1 μg of total RNA from each sample was reverse transcribed by Takara RT reagent Kit (TaKaRa, Dalian, China). Quantitative real-time PCR (qPCR) was performed on the LightCycler® 480 Real-Time PCR Detection System (Roche, Basel, Switzerland) with SYBR Green Kits (Takara, Dalian, China). Gene specific primer sequences were designed by Primer 5.0 (Supplementary Materials, Table S3 ). Amplification conditions were 95 °C for 30 s, followed by 40 cycles of 95 °C for 10 s, 58 °C for 20 s and 72 °C for 10 s. The housekeeping gene gapdh was used as an internal control for normalization based on our previous study [75 (link)]. The 2–ΔΔCt method was used to calculate the relative expression ratio [82 (link)]. All experiments were performed in three biological replicates and two technical replicates.
5
Quantifying Transcript Levels via RT-qPCR
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Total RNA isolated from tissue and cell samples using Trizol reagent (Invitrogen, Life Technologies, USA) were reverse-transcribed to cDNAs using the TaKaRa RT reagent kit (TaKaRa, Shiga, Japan). The LINC00152 and SIRT2 expression was analyzed using SYBR-PCR detection system (Applied Biosystems, USA). The PCR primers used are provided in Table 2 .
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