Peripheral venous blood was collected from patients into EDTA-containing tubes and centrifuged at 3000 × g for 10 min at 4 ℃, and plasma was stored at −80 ℃. Total RNA was extracted from 200 µl plasma samples by using the miRNeasy Serum/Plasma Kit (Qiagen, Hilden, Germany) and cDNA was synthesized using a reverse transcription kit (RiboBio, Guangzhou, China) according to the manufacturer’s instructions. After equal volume dilution of cDNA (20 μl of DNase/RNase-Free Deionized water was added to 20 µl of cDNA), the expression levels of miR-30c-1-3p, miR-432-3p, miR-3154, and miR-379-5p were evaluated by quantitative real-time polymerase chain reaction (qRT-PCR) using specific primers (Supplemental Table 3 ) and the miDETECT A TrackTM miRNA qRT-PCR Kit (RiboBio) following the manufacturer’s protocol; reactions were performed on a CFX96 Touch™ Real-Time PCR Detection System (Hercules, CA, USA). Each reaction was performed in triplicate, and the relative expression level of miRNAs were calculated based on cycle threshold (Ct) values through formula 2-∆Ct, which ∆Ct value is the Ct value of miRNAs in each patient of URAAA/RAAA group minus the average Ct value of miRNAs in the control group.
Midetect a track mirna qrt pcr kit
Manufactured by RiboBio
Sourced in China
The MiDETECT A Track miRNA qRT-PCR Kit is a laboratory equipment product designed for the detection and quantification of microRNA (miRNA) expression using real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) technology. The kit provides a comprehensive solution for miRNA analysis, including reagents and protocols for reverse transcription, real-time PCR amplification, and data analysis.
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Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (6 mentions)
Bulge loop mirna qrt pcr kit, by RiboBio (2 mentions)
U6 small nuclear rna rnu6b, by RiboBio (2 mentions)
Sybr premix ex taq, by Takara Bio (2 mentions)
Revertaid first strand cdna synthesis kit, by Thermo Fisher Scientific (2 mentions)
Primescript rt master mix, by Takara Bio (2 mentions)
Nanodrop 1000 spectrophotometer, by Thermo Fisher Scientific (2 mentions)
Reverse transcription kit, by RiboBio (1 mentions)
Mirneasy serum plasma kit, by Qiagen (1 mentions)
Sybr green qrt pcr, by Takara Bio (1 mentions)
Cfx96 real time detection system, by Bio-Rad (1 mentions)
Itaq universal sybr green supermix kit, by Bio-Rad (1 mentions)
Abi 7500 system, by Thermo Fisher Scientific (1 mentions)
Nanodrop 2000c spectrophotometer, by Thermo Fisher Scientific (1 mentions)
Rnaiso plus, by Takara Bio (1 mentions)
Sybr green qpcr kit, by Thermo Fisher Scientific (1 mentions)
Applied biosystems viia 7 real time pcr system, by Thermo Fisher Scientific (1 mentions)
Trizol reagent, by Takara Bio (1 mentions)
Primescript rt reagent kit, by Takara Bio (1 mentions)
Thunderbird sybr qpcr mix, by Toyobo (1 mentions)
14 protocols using midetect a track mirna qrt pcr kit
1
Plasma miRNA Expression Analysis
2
Quantitative Analysis of mRNA and miRNA
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Total RNA was extracted from the cells and tissues using Trizol method. The purity and concentration of RNA were determined by ultraviolet spectrophotometry. RNA integrity was assessed by agarose gel electrophoresis. Total RNA was used to synthesize cDNA by reverse transcription with the SuperScript™ RT reagent Kit (Thermo Fisher Scienti c, Waltham, USA) with a reaction system volume of 20μL. Expression of the mRNAs was evaluated using SYBR green qRT-PCR (Takara Biotechnology Ltd., Dalian, China) under the standard protocol. GAPDH was used as an internal control, and the relative quantitative method was applied to calculate the relative mRNA copy number (measured in triplicate) 2 -ΔΔCt was used to express the ratio of target mRNA expression relative to the GAPDH mRNA expression. The miDETECT A TrackTMmiRNA qRT-PCR kit (RiboBio, Guangzhou, China) was used to synthesize the cDNA of miR-126 and analyze its expression. U6 small nuclear RNA was used as an internal control for assay of miRNA expression levels, and expression of each gene was quanti ed by measuring cycle threshold (Ct) values and normalized using the 2 -∆∆Ct method relative to U6 small nuclear RNA 16 . The RT-PCR primers are listed in Supplementary Table 1.
3
Gonad Sex-Specific miRNA and RNA Profiling
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Dissected left or right gonads from embryonic stage 29 were pooled according to sex, including 3 biological replicates. Approximately 50 gonads were used for each pool. Total RNA was isolated by using the Trizol reagent (Invitrogen Inc., Carlsbad, CA) according to manufacturer's protocol. The levels of miRNAs were measured by quantitative real-time PCR (qRT-PCR ) using miDETECT A Track miRNA qRT-PCR Kit (RiboBio, Guangzhou, China) according to the manufacturer's instructions. The primers for gga-miR-2954, gga-miR-202-5p, gga-miR-302b-3p, gga-miR-302b-5p, gga-miR-302d, and U6 small nuclear RNA were obtained from RiboBio Company (Guangzhou, China). The miRNA expression levels were normalized to the expression of the internal control U6 using the 2−ΔΔCT. The mRNA and lncRNA template were reversely transcribed into cDNA using a FastQuant RT Kit according to the manufacturer's protocol. qRT-PCR was carried out using iTaq Universal SYBR Green Supermix kit (Bio-RAD, Berkeley, CA) in Bio-RAD CFX96 Real-Time Detection system. Samples were normalized against HPRT using the comparative CT method (ΔΔCT) as previously described (Smith et al., 2008 (link), Ayers et al., 2013 (link)). The primers sequences of mRNAs and lncRNAs used in this study are listed in Supplementaty Table 1 .
4
Quantifying miRNA Expression in PBMCs and RPE
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Total RNA, including miRNA, was extracted from unstimulated PBMCs and transfected RPE cells using TRIzol reagent (Invitrogen, USA). RNA concentration was assessed by NanoDrop 2000 (Wilmington, DE). The levels of miRNAs were measured by qRT-PCR using miDETECT A Track™ miRNA qRT-PCR Kit (RiboBio, Guangzhou, China) and performed on ABI 7500 System (Applied Biosystems). The primers for miR-23a, miR-301a and U6 small nuclear RNA were obtained from RiboBio Company (Guangzhou, China). The sequences are covered by a patent. Analyses of miRNA expression were normalized to the expression of internal control U6 using the 2−ΔΔCT method.
5
Quantitative Analysis of Retinal Gene Expression
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Total RNA was extracted from the retinal cells of patients with age-related cataracts and glaucoma. First-strand cDNA quantitative real-time PCR was performed using 1 μg of total RNA, an oligo primer, the UEIris II RT-PCR System, and a First-Strand cDNA Synthesis Kit (UElandy, Suzhou, China) according to the manufacturer's instructions. To detect gene expression, 2 × SYBR Green qPCR Master Mix (UElandy, Suzhou, China.) with 50 ng of cDNA as a template was analyzed using the Multicolor Real-Time PCR Detection System (Roche Lightcycler 480; Basel, Switzerland). The specific primers are listed in Table S2 and were synthesized by Sangon Biotech (Shanghai, China). The primers for Ier2 and miR-1839 were provided by RiboBio Biotechnology (Guangzhou, China). The levels of miRNAs were measured via qRT-PCR using the miDETECT A Track™ miRNA qRT-PCR Kit, which contains an miRNA-specific forward primer and U6 primer (RiboBio, Guangzhou, China).
6
Quantitative Gene Expression Analysis
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Quantitative RT-PCR was conducted as described previously.28 The primers used for GAPDH, Arg1 (The result of agarose gel electrophoresis for ARG1 was shown in Supplementary Figure 1 Supplementary Table 1
7
Quantitative Gene and miRNA Expression Analysis
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Cells were lysed in RNAiso Plus (Takara) for total RNA extraction. The quantity and quality of RNAs were examined using NanoDrop™ 2000 c spectrophotometers, and 500 ng of RNA of each sample was subjected to reverse transcription to complementary DNA (cDNA) using the RevertAid First-Strand cDNA Synthesis Kit (Thermo Scientific). Quantitative PCR was performed on a 7900 HT Fast Real-Time PCR System with results normalized to the β-actin. The ΔΔCT method was used to calculate relative expression. Primer sequences used in quantitative PCR are shown in Supplementary Table S1 .
For miRNA expression analysis, RNA was reverse transcribed using a miDETECT A Track miRNA qRT-PCR Kit and a Bulge-Loop miRNA qRT-PCR Kit (RiboBio) according to the manufacturer's instructions. Expression levels were normalized to U6-snRNA expression. All experiments were performed in triplicate.
For miRNA expression analysis, RNA was reverse transcribed using a miDETECT A Track miRNA qRT-PCR Kit and a Bulge-Loop miRNA qRT-PCR Kit (RiboBio) according to the manufacturer's instructions. Expression levels were normalized to U6-snRNA expression. All experiments were performed in triplicate.
8
Quantification of miR-141 and its transcripts
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Total RNA was isolated from tissue samples and cell lines using TRIzol reagent (Invitrogen) according to the manufacturer’s protocol. The levels of mature miR-141 (miR-141) and precursor miR-141 (pre-miR-141) were evaluated using the miDETECT A Track miRNA qRT-PCR Kit (RiBoBio). The U6 small nuclear RNA (RNU6B) (RiBoBio) was used for normalization. The expression of primary miR-141 transcript (pri-miR-141), c-Myc and BRD7 was measured by qRT-PCR according to the instructions of the SYBR Premix Ex Taq (TaKaRa, Dalian, China). The GAPDH mRNA level was used for normalization. The relative expression ratio was calculated using the 2−ΔΔCT method. The primers for miR-141, pre-miR-141, U6, pri-miR-141, c-Myc, BRD7, and GAPDH were described previously [23 (link)]. PCRs of each sample were conducted at least in triplicate.
9
Quantitative Analysis of mRNA and miRNA Expression
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Total RNA was isolated from the cells and tissues using TRIzol reagent (Takara). Reverse transcription was conducted with the reverse transcription kit and qPCR kit (Takara). PCR was carried out using the SYBR Green qPCR Kit (Thermo Fisher Scientific, Inc.) with Applied Biosystems Viia7 Real-Time PCR System (Thermo Fisher Scientific, Inc.). Relative expression levels of mRNA were normalized to β-actin and calculated with the 2−ΔΔCq method. Primers of mRNA used in this study are listed in Table SI . The levels of miRNAs were measured by RT-qPCR using miDETECT A Track miRNA qRT-PCR Kit (RiboBio Co.). The primers for miRNAs and U6 small nuclear RNA were obtained from RiboBio Co. The sequences are covered by a patent. Analyses of miRNA expression were normalized to the expression of internal control U6 using the 2−ΔΔCq method.
10
Quantification of miRNA Expression Levels
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Total RNA was isolated from tissue samples and cell lines using TRIzol reagent (Invitrogen) according to the manufacturer's protocol. The expression level of mature miR-141 and pre-miR-141 was evaluated using the miDETECT A Track miRNA qRT-PCR Kit (RiBoBio) following the manufacturer's protocol. The U6 small nuclear RNA (RNU6B) (RiBoBio) was used for normalization. The expression level of pri-miR-141 was measured by qRT-PCR according to the instructions of the SYBR Premix Ex Taq (TaKaRa, Dalian, China). The GAPDH mRNA level was used for normalization. The relative expression ratio of mature miR-141, pre-miR-141 and pri-miR-141 was calculated using the 2−ΔΔCT method. The primer for mature miR-141 was 5′-TAACACTGTCTGGTAAAGATGG-3′ (forward). Primer for pre-miR-141 was 5′-TTGGATGGTCTAATTGTGAAGCTCC-3′ (forward). Primer pairs for U6 were 5′-ATTGGAACGATACAGAGAAGATT-3′ (forward) and 5′-GGAACGCTTCACGAATTTG-3′ (reverse). Primer pairs for pri-miR-141 were 5′-AGACCTCACCTGGCCTGTGGCC-3′ (forward) and 5′-GAACCCACCCGGGAGCCATCTT-3′ (reverse). Primer pairs for GAPDH were 5′-CGAGATCCCTCCAAAATCAA-3′ (forward) and 5′-TTCACACCCATGACGAACAT-3′ (reverse). PCRs of each sample were conducted in triplicate.
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