Total RNA from kidneys samples was reverse-transcribed using miRNA-specific stemloop RT primers, RTase, RT buffer, dNTPs, and RNase inhibitor according to the manufacturer’s instructions (Applied Biosystems; ABI, Foster City, CA). Expression level of miR-146a was quantified using real-time reverse transcription-PCR with the Taqman chemistry (ABI) and miRNA-specific TaqMan primers (Table 1 ), as described previously45 (link). U6 small nuclear RNA was used as endogenous housekeeping control for data normalization of miRNA levels.
Mirna specific stem loop rt primers
Manufactured by Thermo Fisher Scientific
Sourced in United States
The MiRNA-specific stem-loop RT primers are a set of oligonucleotides designed to detect and quantify specific microRNA (miRNA) molecules. These primers facilitate the reverse transcription (RT) of miRNA into complementary DNA (cDNA), which can then be used in downstream applications such as real-time PCR analysis.
Automatically generated - may contain errors
Lab products found in correlation
Taqman microrna assay, by Thermo Fisher Scientific (4 mentions)
Taqman microrna reverse transcription kit, by Thermo Fisher Scientific (3 mentions)
Rnase inhibitor, by Thermo Fisher Scientific (2 mentions)
Taqman mirna assay kit, by Thermo Fisher Scientific (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
7500 real time pcr system, by Thermo Fisher Scientific (1 mentions)
Sybr premix ex taq 2 kit, by Takara Bio (1 mentions)
Reverse transcriptase, by Thermo Fisher Scientific (1 mentions)
Dntps, by Thermo Fisher Scientific (1 mentions)
Rt buffer, by Thermo Fisher Scientific (1 mentions)
Taqman universal pcr master mix, by Thermo Fisher Scientific (1 mentions)
Mirneasy kit, by Qiagen (1 mentions)
Mx3000p, by Agilent Technologies (1 mentions)
Cfx96 real time pcr detection system, by Bio-Rad (1 mentions)
Steponeplus system, by Thermo Fisher Scientific (1 mentions)
7 protocols using mirna specific stem loop rt primers
1
Quantifying miR-146a Expression in Kidney
2
Quantitative miRNA Expression Analysis
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Total RNA was extracted from the tissues and MCCs using TRIzol Reagent (Invitrogen). For quantitative detection of miRNA, a TaqMan miRNA assay kit (Thermo Fisher, USA) was used. Purified miRNA was reverse transcribed using miRNA-specific stem-loop RT primers (Applied Biosystems, USA). Following the manufacturer’s instructions, reverse transcription–quantitative PCR (RT-qPCR) was performed in a 7500 Real-Time PCR system (Applied Biosystems, USA) using SYBR® Premix Ex Taq II Kit (TaKaRa, Japan). Gene expression was normalized to U6 small nuclear RNA expression. The relative gene expression was measured by using the comparative threshold cycle (2−ΔΔCt) method, and β-actin served as an internal control. The reaction mixtures were incubated at 95 °C for 10 min, followed by 40 cycles of 20 s at 95 °C and 60 s at 55 °C. The primers used are shown in Supplementary 4 (the primer sequence of IL-1 β was supplemented in Supplementary 4 ).
3
Quantification of Mature miRNA Expression
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Total RNA (20 ng) and miRNA-specific stem-loop RT primers (Applied Biosystems) were used for the reverse transcription reactions of miRNA. The cDNAs were then synthesized with the TaqMan MicroRNA Reverse Transcription Kit (Applied Biosystems) according to the manufacturer’s protocol. RT-qPCR was performed with TaqMan MicroRNA assays (Applied Biosystems) to analyze the expression of the following mature miRNAs: miR-423 (assay ID: 465126), miR-494 (assay ID: 462468), miR-20a (assay ID: 000580), and miR-17 (assay ID: 002308), selected based on the highest target and rank scores in the miRDB database for miRNA target prediction and functional annotations (http://mirdb.org , accessed date: 16 December 2020). The relative amount of each miRNA was assessed using the comparative CT method (2−ΔΔCt) and normalized to the U6 small nuclear RNA (U6 snRNA).
4
Quantifying miRNA Expression in Glomeruli and Serum
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MicroRNA (miRNA) expression was analysed as described previously19 (link). Briefly, total RNA including miRNA in the glomeruli and serum was isolated using an miRNeasy kit (Qiagen). Total RNA was reverse-transcribed using miRNA-specific stem-loop RT primers, reverse transcriptase, RT buffer, dNTPs, and RNase inhibitor according to the manufacturer's instructions (Applied Biosystems, Foster City, CA, USA). Real-time PCR was performed with the resulting cDNA using miR-21-specific TaqMan primers with specific probes (Applied Biosystems), a TaqMan Universal PCR Master Mix (Applied Biosystems), and Mx3000P (Agilent Technologies).
5
Quantitative Analysis of miRNA Expression
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Total RNA (20 ng) and miRNA-specific stem-loop RT primers (Applied Biosystems) were used for miRNA reverse transcription reactions. The cDNA was then synthesized with TaqMan MicroRNA Reverse Transcription Kit (Applied Biosystems) according to the manufacturer’s protocol RT-qPCR was performed using TaqMan MicroRNA assays (Applied Biosystems) to analyze the expression of the following mature miRNAs: miR-544 (assay ID: 463542) and miR-374-5p (assay ID: 001319) selected from the highest target and ranking scores in the miRDB database for miRNA target prediction and functional annotation (http://mirdb.org , accessed on 16 December 2020). The relative amount of each miRNA was estimated using the comparative CT method (2−ΔΔCt) and normalized to U6 small nuclear RNA (U6 snRNA).
6
Quantitative Analysis of miRNA Expression
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The total RNA (20 ng) and miRNA-specific stem-loop RT primers (Applied Biosystems, USA) were used for the reverse transcription reactions. The cDNAs were then synthesized with the TaqMan® MicroRNA Reverse Transcription kit (Applied Biosystems, USA) according to the manufacturer’s protocol.
Real-time PCR was performed with TaqMan® MicroRNA assays (Applied Biosystems, USA) to analyze the expression of the following mature miRNAs: miR-124, miR-132, miR-134, and miR-212. The 15-μl reaction mixture contained 7.5 μl of 2× Universal PCR Master Mix, 0.75 μl of TaqMan MicroRNA assay, 1.75 μl H2O, and 5 μl of the RT product (diluted 1:15). Each reaction was run in duplicate in a 96-well plate with the following thermal conditions: 95 °C for 10 min, followed by 40 cycles of 15 s at 95 °C and 60 s at 60 °C. All PCR reactions were performed in a Bio-Rad CFX96 Real Time PCR Detection System, and the data were analyzed using CFX Manager Software 2.1. The relative amount of each miRNA was assessed using the comparative CT method (2−ΔΔCt) and normalization to the U6 small nuclear RNA (U6 snRNA). Because even subtle changes in the miRNA levels could have profound effects on their mRNA targets [32 , 33 ], a fold-change cutoff of ≥1.3 (p < 0.05) was applied.
Real-time PCR was performed with TaqMan® MicroRNA assays (Applied Biosystems, USA) to analyze the expression of the following mature miRNAs: miR-124, miR-132, miR-134, and miR-212. The 15-μl reaction mixture contained 7.5 μl of 2× Universal PCR Master Mix, 0.75 μl of TaqMan MicroRNA assay, 1.75 μl H2O, and 5 μl of the RT product (diluted 1:15). Each reaction was run in duplicate in a 96-well plate with the following thermal conditions: 95 °C for 10 min, followed by 40 cycles of 15 s at 95 °C and 60 s at 60 °C. All PCR reactions were performed in a Bio-Rad CFX96 Real Time PCR Detection System, and the data were analyzed using CFX Manager Software 2.1. The relative amount of each miRNA was assessed using the comparative CT method (2−ΔΔCt) and normalization to the U6 small nuclear RNA (U6 snRNA). Because even subtle changes in the miRNA levels could have profound effects on their mRNA targets [32 , 33 ], a fold-change cutoff of ≥1.3 (p < 0.05) was applied.
7
Quantifying miR-615 Expression using RT-qPCR
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We measured miR-615 expression in 77 available study samples using TaqMan MicroRNA Assays (Applied Biosystems) according to the manufacturer's guidelines. Briefly, miRNAs were reverse transcribed using miRNAspecific stem-loop RT primers (Applied Biosystems) and 10 ng RNA input for each 15 μL RT reaction and 1.33 μL cDNA input for each 20 μL PCR reaction (Applied Biosystems StepOnePlus System). PCR reactions were incubated in a 96-well plate format at 95°C for 10 min, followed by 40 cycles at 95°C for 15 s and 60°C for 1 min. All samples were assayed in triplicate. Mean Ct values were calculated for each sample and normalized against the corresponding U6 Ct values, calculated as -(Ct_miR-Ct_U6) (Schmittgen & Livak 2008) (link); all data are presented as normalized Ct values. To assess the degree of similarity between RT-qPCR and sequencing results, we compared and correlated miR-615 expression data generated through both approaches.
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