Total RNA was extracted from lungs and cells using RNAiso Plus (TaKaRa, Tokyo, Japan). The isolated total RNA was reverse transcribed using Mir-X miRNA First-Stand Synthesis Kit (TaKaRa, Tokyo, Japan) for microRNA let-7d and the PrimerScript™ RT Reagent Kit with gDNA Eraser (TaKaRa, Tokyo, Japan) for mRNA, according to manufacturer instructions. Relative expression was assessed by ABI7500 Fast Real-Time PCR System (Applied Biosystems, USA) with the TB Green Premix Ex II (TaKaRa, Tokyo, Japan). Relative expression was calculated using the 2−ΔΔCT method and was normalized to the expression of U6 or GAPDH. ALL qRT-PCR reactions were performed in triplicate. The sequences of primer pairs are described in Table 1 .
Mir x mirna first stand synthesis kit
Manufactured by Takara Bio
Sourced in Japan, China
The Mir-X miRNA First-Strand Synthesis Kit is a laboratory equipment product designed for the conversion of mature microRNA (miRNA) into first-strand complementary DNA (cDNA). This kit provides the necessary reagents and protocols for the reverse transcription of miRNA into cDNA, which can then be used for downstream applications such as real-time PCR analysis.
Automatically generated - may contain errors
Lab products found in correlation
Rnaiso plus, by Takara Bio (1 mentions)
Primerscript rt reagent kit with gdna eraser, by Takara Bio (1 mentions)
Abi 7500 fast real time pcr system, by Thermo Fisher Scientific (1 mentions)
Tb green premix ex 2, by Takara Bio (1 mentions)
Mirneasy micro kit, by Qiagen (1 mentions)
Cfx96 real time detection system, by Bio-Rad (1 mentions)
Nanodrop spectrophotometer, by Thermo Fisher Scientific (1 mentions)
Tb green premix ex taq, by Takara Bio (1 mentions)
Mirna specific primer, by RiboBio (1 mentions)
Sybr premix ex taq, by Takara Bio (1 mentions)
Cfx96 detection system, by Bio-Rad (1 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (1 mentions)
Primescript rt reagent kit, by Vazyme (1 mentions)
Trizol, by Thermo Fisher Scientific (1 mentions)
Methanol, by Merck Group (1 mentions)
Anti creb, by Abcam (1 mentions)
Formic acid, by Merck Group (1 mentions)
Anti cx43, by Abcam (1 mentions)
Sybr premix dimer eraser, by Vazyme (1 mentions)
Gapdh, by Wuhan Sanying Biotechnology (1 mentions)
6 protocols using mir x mirna first stand synthesis kit
1
Quantitative Analysis of miRNA and mRNA
2
Exosomal miRNA Isolation and Quantification
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miRNeasy Micro Kit (QIAGEN, Valencia, CA, USA) was used to isolate exosomal total RNA. Exosome samples were processed according to the manufacturer's instruction as described in a previous study (35 (link)). The extracted RNA was resuspended with 16 μl of nuclease-free water. Then we used NanoDrop spectrophotometer (Thermo Fisher Scientific, Lafayette, USA) to evaluate the concentration and quality of the RNA. cDNA was synthesized from 200 ng of template RNA using the Mir-X MiRNA First-Stand Synthesis Kit (Takara, Dalian, China) in a 10 μl of reaction volume.
After 5-fold dilution, 2 μl of cDNA was used for qPCR assay that was reacted using the TB Green™ Premix Ex Taq™ (Takara, Dalian, China) on a CFX96 Real-Time Detection System (Bio-Rad Laboratories, USA). MiRNA primers were synthesized by Ribobio (Guangzhou, China), and forward primer sequence has been provided inSupplementary Table 1 . Each qRT-PCR was repeated three times. The relative expression of miRNAs was calculated and normalized to miR-16, using the comparative threshold (2−ΔΔCt) method.
After 5-fold dilution, 2 μl of cDNA was used for qPCR assay that was reacted using the TB Green™ Premix Ex Taq™ (Takara, Dalian, China) on a CFX96 Real-Time Detection System (Bio-Rad Laboratories, USA). MiRNA primers were synthesized by Ribobio (Guangzhou, China), and forward primer sequence has been provided in
3
Quantitative PCR Analysis of miRNA
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Briefly, 2 μg of total RNA was reversely transcribed into cDNA using the Mir-X miRNA First-Stand Synthesis Kit (Takara, Shiga, Japan). Then, 2 μL of cDNA was used for quantitative PCR analysis that was performed on the Bio-Rad CFX96 Detection System (Bio-Rad, Hercules, CA) using the SYBR Premix EX Taq (Takara, Shiga, Japan) and miRNA-specific primers (Ribobio, Guangzhou, China). All reactions were performed in triplicate to remove any outliers. In addition, miR-191-5p and U6 were selected as the reference genes according to our previous study [31 ]. The miRNA expression was normalized to the mean of these two reference genes. The relative expressions of miRNAs were determined using the 2-dCt method.
4
Mahonia-Based Antidepressant Protocol
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Dried Mahonia were purchased form Guangxi Xianju Chinese Medicine Technology Co.. Acetonitrile (Nanning, Guangxi, China); methanol and formic acid were provided by Sigma (Sigma-Aldrich, USA); Fluoxetine was supplied by Lilly Suzhou Pharmaceutical Co., Ltd. (Suzhou, Jiangsu, China); The enzymelinked immunosorbent assay (ELISA) kits for rat and human MAO, DA, NE, 5-HT were supplied by Shanghai Fanke Industrial Co., Ltd. (Shanghai, China); Lipofectamine 3000 and Trizol were provided by Invitrogen (USA); The PrimeScript RT Reagent Kit and SYBR Premix Dimer Eraser were provided by Vazyme Biotech Co., Ltd, (Nanjing, Jiangsu, China); The Mir-x miRNA First-Stand Synthesis Kit was purchased from TaKaRa (Japan); Anti-Cx43 and Anti-CREB were provided by Abcam (UK), Anti-p-CREB, Anti-BDNF, GAPDH were provided by Wuhan Sanying Biotechnology Co., Ltd. (Wuhan, Hebei, China); Corticosterone (CORT) was provided by Aladdin (Shanghai, China).
5
Serum Total RNA Extraction and miRNA qRT-PCR
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Total RNA was obtained from serum using Trizol LS Reagent (Invitrogen, Carlsbad, California, USA) following the manufacturer's instructions. The optical density values were detected using an ultraviolet spectrophotometer with A260/A280 ranging from 1.8 to 2.1. Reverse transcription was carried out with the Mir‐X miRNA First‐stand Synthesis Kit (Clontech Laboratories, Mountain View, California, USA) at 37°C for 60 min and then at 85°C for 5 min. The complementary deoxyribonucleic acid (cDNA) was diluted to 100 μL for further study. Quantitative reverse transcription polymerase chain reaction (qRT–PCR) was carried out by Mir‐X miRNA qRT–PCR SYBR Kit (SYBR Green; Clontech Takara, Otsu, Shiga, Japan) using the Roche Light Cycler Fluorescence Quantitative PCR System (ABI, Waltham, Massachusetts, USA) with 2 μL cDNA as the template under the following conditions: 95°C for 10 s, 40 cycles at 95°C for 5 s and 54°C for 20 s. All the PCR reactions were in a reaction volume of 25 μL and repeated three times. The results were represented as cycle threshold values.
6
Quantifying miRNA-322 Expression in Heart Tissue
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