Specific paired primers for qRT-PCR were designed and synthesized by Sangon Biotech (Supplementary Table S1). Universal reverse primers and U6 primers were provided by the Mir-X miRNA First-Strand Synthesis Kit (TaKaRa, Japan), and used for miRNA expression analysis. cDNAs were synthesized from total RNAs with a cDNA synthesis kit (TaKaRa) or the Mir-X miRNA First-Strand Synthesis Kit. Subsequently, qRT-PCR amplification was performed via SYBR Green PCR Premix Ex TaqTM II reagents (TaKaRa) with the QuantStudio 6 FlexI real-time system (Applied Biosystems, U.S.A.) following the protocol of this product. The levels of targeted genes were determined with the 2(−ΔΔCt) method in comparison with endogenous controls (GAPDH or U6).
Mir x mirna first strand synthesis kit
Manufactured by Takara Bio
Sourced in Japan, China, United States
The Mir-X miRNA First-Strand Synthesis Kit is a tool designed for the reverse transcription of mature microRNA (miRNA) molecules. The kit contains the necessary reagents and protocols to convert miRNA into cDNA, which can then be used for downstream applications such as real-time PCR or other miRNA analysis techniques.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (205 mentions)
Primescript rt reagent kit, by Takara Bio (89 mentions)
Trizol, by Thermo Fisher Scientific (61 mentions)
Primescript rt reagent kit with gdna eraser, by Takara Bio (51 mentions)
Tb green premix ex taq 2, by Takara Bio (50 mentions)
Sybr premix ex taq 2, by Takara Bio (48 mentions)
Primescript rt master mix, by Takara Bio (46 mentions)
Mir x mirna qrt pcr tb green kit, by Takara Bio (42 mentions)
Mirneasy mini kit, by Qiagen (34 mentions)
Nanodrop 2000, by Thermo Fisher Scientific (34 mentions)
Trizol reagent, by Takara Bio (31 mentions)
Cfx96 real time pcr detection system, by Bio-Rad (28 mentions)
Mir x mirna qrt pcr sybr kit, by Takara Bio (28 mentions)
Steponeplus real time pcr system, by Thermo Fisher Scientific (26 mentions)
Sybr premix ex taq, by Takara Bio (25 mentions)
Rnaiso plus, by Takara Bio (23 mentions)
Sybr advantage qpcr premix, by Takara Bio (17 mentions)
Sybr premix ex taq kit, by Takara Bio (17 mentions)
Sybr premix ex taq 2 kit, by Takara Bio (17 mentions)
Revertaid first strand cdna synthesis kit, by Thermo Fisher Scientific (17 mentions)
651 protocols using mir x mirna first strand synthesis kit
1
Quantitative Real-Time PCR Protocol
2
Quantify miR-4469 Expression in Cells
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Total RNA in cells was extracted using Trizol (TaKaRa, Japan). Mir-X™miRNA First Strand Synthesis Kit (TaKaRa, Japan) was used to reverse transcribe miRNA into cDNA. A quantitative polymerase chain reaction (qPCR) was conducted using the 7500 Real-Time PCR Systems (Applied Biosystems, USA) with TB Green®Premix Ex Taq™II (TaKaRa, Japan) as the reaction reagent. The upstream miR-4469 primer sequence was 5ʹ-CTCTAGGGTCGCTCGGAAA-3ʹ. The upstream and downstream primers of U6 and the downstream primer of miR-4469 were derived from the Mir-X™miRNA First Strand Synthesis Kit (TaKaRa, Japan).
3
Quantitative Analysis of miRNA Expressions
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Total RNA samples were extracted from treatments in the use of TRIzol reagent kit (Invitrogen, United States), according to the instruction of the manufacturer, and the quality of all the RNA samples was assessed via agarose gel electrophoresis, and also, an ultraviolet spectrophotometer (Nano-Drop-2000, Thermo Scientific) was utilized to check the concentration and purity of the RNA samples, according to the published method (Yang et al., 2017 (link)). Then, with the use of RNA as a template, the steps of reverse transcription were carried out following the specification of the Mir-X miRNA First-Strand Synthesis Kit (TaKaRa, China). A total of 10 miRNAs (miR-1-3p, miR-9a, miR-11, miR-184, miR-100-5p, miR-275, miR-252a, miR-279d, miR-277, and miR-624) and U6 snRNA, one commonly used reference gene, were set as the candidates for evaluation, according to the published database of B. tabaci miRNA-seq and our database (Wang et al., 2016 (link); Li et al., 2018 (link); Hasegawa et al., 2020 (link)). Primers of the candidate miRNA were designed in utilizing the software of Primer 5.0 based on the specification of the Mir-X miRNA First-Strand Synthesis Kit (TaKaRa, China), and the reverse primer of all miRNAs was a universal reverse primer (mRQ 3′Primer) from the kit.
4
Validating RNA-seq Data with miRNA
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Seven DEMs were selected to validate the accuracy of the RNA-seq, including 5 common DEMs in both comparison groups. cDNA synthesis was performed using Mir-X miRNA First-Strand Synthesis Kit (Takara Bio, Beijing, China). Forward primers for all seven miRNAs were designed through miRNA Design V1.01 (Table S1 ). Forward and reverse primers of housekeeping gene U6 and universal reverse primers were all from Mir-X miRNA First-Strand Synthesis Kit (Takara Bio, Beijing, China). The melting point of primers was 60 °C. Each primer was verified, and the melting curves had a smooth single peak. Calculation of the relative expression of DEMs was performed using the 2-ΔΔCT algorithm.
5
Quantitative Analysis of miR-374b-5p in CD4+ T Cells
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Total RNA was extracted from CD4+T cells of spleen using Trizol reagent (Invitrogen) according to the manufacturer's procedures. For miRNA-specific reverse transcription, the reaction was performed using the Mir-X™ miRNA First-Strand Synthesis Kit (Takara) and reverse transcription primers from Mir-X™ miRNA First-Strand Synthesis Kit (Takara) on the ABI 7500 fast real-time PCR system (Applied Biosystems, Foster City, CA, USA), according to the manufacturer's instructions. The miR-374b-5p PCR primers were purchased from Qiagen (218300). U6 was utilized as an internal control. U6, forward: 5′-CTCGCTTCGGCAGCACA-3′; reverse: 5′-AACGCTTCACGAATTTGCGT-3′. The relative miRNA levels were calculated using the 2 –ΔΔCt method.
6
Quantitative Analysis of RNA Levels
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Total RNA from the cell was obtained using the RNAiso Plus Reagent (Invitrogen, Hong Kong, China) according to the standard guidelines. The Agilent 2100 Bioanalyzer system (Agilent Technologies, Carlsbad, CA, USA) was used for quality inspection, and only qualified RNA (A160/A180 = 1.6–1.8, concentration > 200 ng/uL) was used for the later trial. Reverse transcription of mRNA and miRNA was performed using the PrimeScript™ RT reagent Kit with gDNA Eraser (Takara, Dalian, China) and the Mir-X™ miRNA First-Strand Synthesis Kit (Takara) following the manufacturer’s protocol, respectively. Subsequently, qRT-PCR was performed in triplicate using the TB Green™ Premix Ex Taq™ II (Takara) on a CFX96 instrument (Bio-Rad, Hercules, CA, USA), and the relative expression levels of mRNA and miRNA were calculated using the 2−ΔΔCt method. The mRQ 3′ primer in the Mir-X™ miRNA First-Strand Synthesis Kit (Takara) was used and served as a reverse primer for miRNA quantification, and U6 was used as an internal reference. Besides, GAPHD was used as an internal reference for mRNA quantification. Detailly, the primer sequences are listed in Table 2 .
7
Quantitative miRNA Expression Analysis
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Total RNA was extracted from 0.3 to 0.5 million cells using the Nucleozol RNA extraction reagent (Takara Bio). Subsequently, 1 μg RNA was reverse transcribed using the Mir-X™ miRNA First-Strand Synthesis Kit (Takara Bio) as per the manufacturer’s protocol. The quantitative RT-PCR reaction was set up with Go Taq qPCR Master Mix (Promega) using a miRNA-specific forward primer and a common reverse primer provided in the Mir-X™ miRNA First Strand Synthesis Kit (Takara Bio). The miRNA-specific forward primers were designed using miRprimer2 (Busk, 2014 (link)). The assay was carried out on QuantStudio 12K Flex Real-Time PCR equipment and software (Thermo Fisher Scientific). Melt/dissociation curves were analyzed before the quantification of miRNAs.
8
Quantitative Real-Time PCR Protocol
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Specific paired primers for qRT-PCR were designed and synthesized by Sangon Biotech (Supplementary Table S1). Universal reverse primers and U6 primers were provided by the Mir-X miRNA First-Strand Synthesis Kit (TaKaRa, Japan), and used for miRNA expression analysis. cDNAs were synthesized from total RNAs with a cDNA synthesis kit (TaKaRa) or the Mir-X miRNA First-Strand Synthesis Kit. Subsequently, qRT-PCR amplification was performed via SYBR Green PCR Premix Ex TaqTM II reagents (TaKaRa) with the QuantStudio 6 FlexI real-time system (Applied Biosystems, U.S.A.) following the protocol of this product. The levels of targeted genes were determined with the 2(−ΔΔCt) method in comparison with endogenous controls (GAPDH or U6).
9
miR-135b-5p Expression Analysis
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The Mir-X miRNA First-Strand Synthesis Kit (Takara Bio), Mir-X miRNA First-Strand Synthesis Kit (Takara Bio), and LightCycler® 96 Instrument (Roche, USA) were applied to perform reverse transcription and quantitative real-time polymerase chain reaction (qRT-PCR), according to the manufacturers' instructions. The primer sequence of miR-135b-5p was 5′-GGAGGCTTTTCATTCCTATGTGA-3′, and the primer was synthesized by Takara Biomedical Technology Co., Ltd. Calculation of relative changes in expression was based on the 2−ΔCT method, and all reactions were performed in triplicate.
10
Quantitative Analysis of miRNA Expression
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The expression changes of randomly selected miRNAs were determined by q-PCR approach. The cDNA was synthesized from miRNAs using the Mir-X™ miRNA First Strand Synthesis Kit (Clontech, Dalian, China) according to the supplier’s protocol. Quantitative PCR was carried out using SYBR Premix Ex Taq II (Takara, Dalian, China) on the CFX96™ Real-Time PCR Detection System (Bio-Rad, CA, USA). U6 snRNA were simultaneously used as internal control genes. The miRNA forward primers were obtained commercially from BGI (Shenzhen, China) and the universal reverse primer for miRNAs was offered by the Mir-X™ miRNA First Strand Synthesis Kit (Clontech, Dalian, China). The primer sequences are provided in Additional file 8 : Table S5. Each miRNA sample was analyzed in triplicate and the 2−ΔΔCt method was used to calculate the relative expression levels of objective miRNAs.
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