Total RNAs were isolated using Trizol reagent (Invitrogen) and then reverse transcribed into cDNA by a reverse transcription reagent kit (Takara, Dalian, China). The relative expression of miRNA was performed using SYBR Green PCR Master Mix (Takara) and detected using the Bio-Rad Real-Time PCR instrument (Bio-Rad). The relative level of miRNA was normalized with U6 and quantitative analysis was calculated using the 2–ΔΔCt method. The primer sequences are shown in Table I .
Reverse transcription reagent kit
Manufactured by Takara Bio
Sourced in China, Japan, United States
The Reverse Transcription Reagent Kit is a comprehensive set of reagents designed for the reverse transcription of RNA to complementary DNA (cDNA). The kit includes all the necessary components, such as reverse transcriptase enzyme, reaction buffer, and dNTPs, to efficiently perform this essential step in various molecular biology applications.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (61 mentions)
Trizol, by Thermo Fisher Scientific (13 mentions)
Nanodrop 2000, by Thermo Fisher Scientific (10 mentions)
Trizol reagent, by Takara Bio (7 mentions)
Abi 7900 real time pcr system, by Thermo Fisher Scientific (4 mentions)
Rneasy mini kit, by Qiagen (4 mentions)
Steponeplus real time pcr detection system, by Thermo Fisher Scientific (4 mentions)
Steponeplus real time pcr system, by Thermo Fisher Scientific (4 mentions)
Sybr green master mix, by Takara Bio (3 mentions)
Sybr green mix kit, by Thermo Fisher Scientific (3 mentions)
Sybr premix ex taq kit, by Takara Bio (3 mentions)
Tb green premix ex taq 2, by Takara Bio (3 mentions)
Sybr green mix, by Takara Bio (3 mentions)
Cfx96 real time pcr detection system, by Bio-Rad (3 mentions)
Sybr green mix, by Thermo Fisher Scientific (3 mentions)
Robo cycler thermocycler, by Takara Bio (2 mentions)
Sybr premix ex taq 2, by Takara Bio (2 mentions)
Rnaiso plus, by Takara Bio (2 mentions)
Abi prism 7500 sequence detection system, by Thermo Fisher Scientific (2 mentions)
Sybr premix ex taqtm perfect real time kit, by Takara Bio (2 mentions)
112 protocols using reverse transcription reagent kit
1
miRNA Expression Analysis by RT-qPCR
2
RNA Extraction and qPCR Analysis
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Total RNA was extracted using TRI reagent (Sigma, USA), and cDNA was synthesized using a reverse transcription reagent kit (TaKaRa, Japan). Amplification was performed using SYBR green qPCR master mix (Biotools, China) and gene-specific primers in a CFX-96 system (Bio-Rad, USA), and values were normalized to those of a housekeeping gene. The qPCR primers are listed in Table S4 .
3
Quantitative RT-PCR Analysis of Macrophage and Skin
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Total RNA from peritoneal macrophage and skin tissues were extracted using the TRIzol reagent (Invitrogen), and the cDNAs were synthesized using the Reverse Transcription Reagent Kit (Takara Bio Inc.) according to the manufacturer's instruction. The resulting cDNAs were amplified with 40 cycles by real-time PCR. Each sample was analyzed three times and normalized to a reference RNA using β-actin as the internal control. Sequences of primers used for real-time PCR to analyze the mouse samples are summarized in Table 1 .
4
Extracting and Quantifying Hypothalamic RNA
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Total RNA was extracted from hypothalamus tissue using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) according to the manufacturer's instructions. Purified RNA samples were resuspended in diethypyrocarbonate water. Then RNA was reverse transcribed in 20 µl total volume using the Reverse Transcription Reagent kit (Takara Bio, Inc., Dalian, China) for 60 min at 42°C and 5 min at 70°C. qPCR was performed in an ABI PRISM 7000HT system (Applied Biosystem; Thermo Fisher Scientific, Inc.) in triplicates using the SYBR Green Master Mix (Takara Bio, Inc.). The primers sequences used are presented in Table I . The PCR conditions were as follows: 15 sec at 95°C, 1 cycle; 5 sec at 95°C and 34 sec at 60°C, 40 cycles. β-actin served as the internal control. The 2−ΔΔCq method was used to calculate the relative mRNA levels (24 (link)).
5
Total RNA Extraction and qRT-PCR Analysis
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Total RNA was separated using a Trizol reagent (Life Technologies, USA) according to the manufacturer’s protocols. RNA concentrations and purity were assessed by the measurement of optical density at 260 and 280 nm. The purified total-RNA was reverse transcribed with reverse transcription reagent kit (Takara, Japan) and quantitative real-time PCR was performed on Stepone Plus system using the SYBR Green Master Mix (Takara, Japan). The fold changes in the genes were analyzed based on comparative ΔΔCt method. The sequences of primers were listed in Additional file 1 : Table S1.
6
Comprehensive RNA Isolation and Analysis
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Total RNA was isolated from heart tissues, hypothalamus tissues, cardiac fibroblasts, and cardiomyocytes using TRIzol reagent (15596018, Invitrogen), as previously described (48 (link)). RNA was isolated from endothelial cells and sorted macrophages using RNeasy Plus Micro Kit (74034, QIAGEN) according to the manufacturer’s instructions. Briefly, total RNA (1 μg) was reverse-transcribed to cDNA using the Reverse Transcription Reagent Kit (Takara Bio Inc., Kusatsu). The resulting cDNA was amplified via 40 cycles of real-time PCR using SYBR Green Mix (Applied Biosystems, Waltham, MA, USA). The mRNA level of each target gene was normalized to endogenous GAPDH expression. The ΔΔCt method was used to evaluate relative expression levels or fold changes. The primer sequences used in our study are listed in table S2.
7
Quantifying UCP2 mRNA Expression
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To measure the mRNA of UCP2, the RNA of cerebral cortex was extracted by Trizol reagent (Invitrogen, Carlsbad, CA, USA). Total RNA was reverse transcribed to cDNA by using a reverse transcription reagent kit (Takara, Otsu, Shiga, Japan) in a Robo-cycler thermocycler. Real-time PCR was performed with the LightCycler 480 SYBR Green I Master kit and the products were detected using an a LightCyler 480 instrument. The sequences of Real-time PCR primers used for mRNA quantification were as follows: 5′-TAGTGCGCACCGCAGCC-3′ and 5′-AGCTCATCTG-GCGCTGCAG-3′ for mouse UCP2 and 5′-TGGTGCCAAAAGGGTCATCTCC-3′ and 5′-GCCAGCCCCAGCATCAAAGGTG-3′ for mouse GAPDH. Relative genomic expression was calculated by the 2−ΔΔCt method66 (link),67 (link).
8
Quantitative Real-Time PCR Analysis of Neuritin Transcript
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Total RNA from the cortex, hippocampus, and U-118MG cells was extracted using RNAiso Plus (TAKARA, Catalog#9108/9109) (Tang et al. 2017 (link)). cDNA was synthesized using a Reverse-Transcription Reagent Kit (TAKARA, Catalog#RR047A) (Tang et al. 2017 (link)). Real-time PCR measurements of individual cDNAs were performed using SYBR Premix Ex Taq™ II (TAKARA, Catalog#RR820A) to measure the duplex DNA formation with the ABI Prism 7500 Sequence Detection System (Applied Biosystems) (Tang et al. 2017 (link)), normalized to the amount of β-actin RNA and analyzed by the 2−∆∆CT method (Livak & Schmittgen 2001 (link)). The following primers were used: β-actin sense 5-CTCTAGACTTCGAGCAGGAGAT-3; β-actin antisense 5-CAGGATTCCATACCCAAGAAGG-3; neuritin sense 5-GCGGTGCAAATAGCTTACCTG-3, neuritin antisense 5-CGGTCTTGATGTTCGTCTTGTC-3′.
9
Quantifying Immune Checkpoint Receptor Expression
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Total RNA was extracted from fresh PBMCs by the RNeasy Mini Kit (Qiagen, Germany), which was used to synthesize cDNA by a reverse transcription reagent kit (Takara, China). Real-time quantitative PCR was used to detect the expression levels of target genes in triplicate by Takara SYBR Supermix (Takara, China) by ABI QuantStudio 5 (Applied Biosystems, United States) as previously described (Shen et al., 2020 (link)). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as an internal control in this study. The sequences of the specific primers are listed as follows: PD-1 forward 5′-AAGGCGCAGATCAAAGAGAGCC-3′ and reverse 5′-CAACCACCAGGGTTTGGAACTG-3′; TIM-3 forward 5′-GACTCTAGCAGACAGTGGGATC-3′ and reverse 5′-GGTGGTAAGCATCCTTGGAAAGG-3′; LAG-3 forward 5′-GCAGTGTACTTCACAGAGCTGTC-3′ and reverse 5′-AAGCCAAAGGCTCCAGTCACCA-3′; CTLA-4 forward 5′-ACGGGACTCTACATCTGCAAGG-3′ and reverse 5′-GGAGGAAGTCAGAATCTGGGCA-3′; and GAPDH forward 5′-GTCTCCTCTGACTTCAAC AGCG-3′ and reverse 5′-ACCACCCTGTTGCTGTAG CCAA-3′.
10
Quantitative Analysis of miRNA and mRNA Expression
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Total RNA was extracted from the clinical specimens using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. Subsequently, the RNAs were reversed transcribed into cDNA using a reverse transcription reagent kit (Takara Biotechnology Co., Ltd., Dalian China). Subsequently, cDNA was amplified using an SYBR Green mix kit and the ABI 7900 Real-Time PCR system (Applied Biosystems; Thermo Fisher Scientific, Inc.) following the manufacturer's instructions. PCR was performed with the following thermocycling conditions: Initial denaturation at 94°C for 4 min, 40 cycles of 94°C for 30 sec, 56°C for 30 sec and 72°C for 25 sec, using the ABI 7900 Real-Time PCR system (Applied Biosystems; Thermo Fisher Scientific Inc.). The primer sequences are shown in Table II . Relative levels of miR-539 and mRNA expression levels of HMGA2 were normalized to that of small nuclear RNA U6 (for miRNAs) or GAPDH (for mRNAs) respectively. The relative expression of miRNA or mRNA was calculated using the 2−ΔΔCt method (15 (link)).
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