Total RNAs were extracted with TRIzol reagent (Invitrogen, Carlsbad, CA). To analyze miR-181a expression, miRNA cDNA Kit (CWBIO, China) and miRNA Real-Time PCR Assay Kit (CWBIO, China) were used. And the miRNA specific forward primer was 5′-AACATTCAACGCTGTCGGTGAGT-3′ and a universal reverse primer was provided by miRNA Real-Time PCR Assay Kit. U6 was used as a miRNA internal control, the primers for U6 were 5′-AGAGCCTGTGGTGTCCG-3′ (forward) and 5′-CATCTTCAAAGCACTTCCCT-3′ (reverse). To measure messenger RNA (mRNA) level of E2F5, total RNA was reversely transcribed using the Reverse Transcription System (Promega, Madison, WI). The primers used were 5′-TCAGGCACCTTCTGGTACACAACT-3′(forward) and 5′- AGCAGCACATGGATAGGTCCTGAA-3′(reverse). β-actin was used as an endogenous control, the primers for it were 5′-GTGGATCAGCAAGCAGGAGT-3′ (forward), 5′-TGTGTGGACTTGGGAGAGGA-3′ (reverse). All quantitative real-time polymerase chain reaction (qRT-PCR) samples were performed by using SYBR Green PCR master mix (CWBIO, China). The real-time PCR reactions were performed in triplicate and included no-template controls. Relative changes in gene expression were calculated using the 2-ΔΔCT method [15 (link)].
Mirna real time pcr assay kit
Manufactured by CWBIO
Sourced in China
The MiRNA Real-Time PCR Assay Kit is a laboratory equipment product designed for the detection and quantification of microRNA (miRNA) expression levels using real-time PCR technology. The kit provides the necessary reagents and protocols for performing miRNA analysis.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (5 mentions)
Ultrasybr mixture, by CWBIO (3 mentions)
Reverse transcription system, by Promega (2 mentions)
Sybr green pcr master mix, by CWBIO (1 mentions)
Mirna purification kit, by CWBIO (1 mentions)
Quantstudio 6 pcr system, by Thermo Fisher Scientific (1 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (1 mentions)
Ultra sybr mixture with rox, by CWBIO (1 mentions)
Piko real 96 pcr system, by Thermo Fisher Scientific (1 mentions)
Biort cdna first strand synthesis kit, by CWBIO (1 mentions)
Dnase, by Promega (1 mentions)
Mirna isolation kit, by CWBIO (1 mentions)
7 protocols using mirna real time pcr assay kit
1
miRNA and mRNA Expression Analysis
2
Validating miRNA-Like RNA Expression
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Experimental validation of miRNA-like RNAs was performed using the miRNA Purification Kit (Cwbio, CW0627), miRNA cDNA Kit (Cwbio, CW2141), and miRNA Real-Time PCR Assay Kit (Cwbio, CW2142). Briefly, a poly-A tail was added to the 3' end of total RNA. Then, the RNA was reverse-transcribed with an oligo-dT adaptor. Quantitative PCR (qPCR) was then performed using Synergy Brands green detection with a forward primer for mature milRNA sequences and a universal adaptor reverse primer.29 To calculate the expression levels of the target gene, the 2−△△CT relative quantification method was employed.30 (link) All reactions were repeated three times with three biological samples and were detected by 0.8% (w/v) agarose gel electrophoresis.
3
Quantification of miR-499a and MAPK6 mRNA
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Total RNA of the cells was extracted with Trizol reagent (Invitrogen). In order to quantitate miR499a expression, miRNA cDNA Kit (CWBIO, China) and miRNA Real-Time PCR Assay Kit (CWBIO, China) were used. U6 was used as a miRNA internal control. To measure the mRNA expression of MAPK6, total RNA was reversely transcribed using the Reverse Transcription System (Promega, Madison, WI) and quantitative real time PCR was performed by using the UltraSYBR Mixture (CWBIO, China). β-Actin were used as an endogenous control. The primers were as follows: MAPK6-F (realtime) (5′-ACTTGGTGCTGAAGATAG-3′ ); MAPK6-R (realtime) (5′-TGAGAAGCTCCTGACGAT-3′ ); β-Actin-F (5′-CCTTCTACAAATGAGCTGCGT-3′ ); β-Actin-R (5′-CCTGGATAGCAACGTA CATG-3′ ). All samples were normalized to internal controls and fold changes were calculated through relative quantification (2−ΔΔct).
4
Extraction and Quantification of CircRNA and miRNA
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A commercial TRIzol reagent (Catalog No: 15596018; Invitrogen, USA) was used to extract the total RNA. For the RNase R assay, the total RNA was treated with 2 U/μg RNase R at 37°C for 20 min. Next, RNA samples were subjected to reverse transcription using the High-Capacity cDNA Reverse Transcription Kit (Catalog No: 4368814; Applied Biosystems, USA) and quantified for RT-qPCR analysis using UltraSYBR Mixture (Catalog No: CW2601M; Cwbio, China). The Quant Studio6 PCR system (Life Technologies, USA) was used for RT-qPCR. For RT-qPCR of miRNAs, cDNA synthesis and quantification were conducted using an miRNA Real-Time PCR Assay Kit (Catalog No: CW2142; Cwbio). All experimental procedures were performed in accordance with the manufacturer’s protocol. In this study, relative expression was identified using the 2−△△Ct method, with GAPDH (for circ_0000285) and U6 (for miR-582-3p) as the housekeeping genes for circRNA and miRNA, respectively. Primer sequences are shown in Table 1 .
5
Quantifying miRNA Expression by RT-qPCR
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We obtained total miRNA from cells by using Trizol reagent (Invitrogen, USA) according to its illustration. RT-qPCR was carried out on a Pikoreal 96 PCR system (Thermo Fisher Scientific, USA). UltraSYBR Mixture (with ROX) (CWBio, China) and the miRNA Real-Time PCR Assay kit (CWBio, China.) were used to detect and measure relative expression of mRNA as well as miRNA, respectively. We normalized the results and analyzed the data based on the 2-ΔΔCt method. The primers in this study were indicated in Table 1 .
6
Quantitative Analysis of miRNA and mRNA
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Total RNA from cell lines containing miRNA was isolated with Trizol reagent (Life Technologies) and treated with DNase (Promega, Madison, WI). First-strand cDNA was generated with a miRNA cDNA Kit or a BioRT cDNA First Strand Synthesis Kit according to the manufacturer's instructions (CWBio, Hangzhou Bioer Technology, China). Quantitative real time polymerase chain reaction (qRT-PCR) was performed using miRNA Real-Time PCR Assay Kit or UltraSYBR mixture (CWBio) to confirm expression of miRNAs or mRNAs. A cycle threshold (CT) was assigned at the beginning of the logarithmic phase of PCR amplification, and duplicate CT values were analyzed using the 2−ΔΔCT method [37 (link)]. U6 and β-actin mRNA were used for normalization. The forward and reverse primers of qRT-PCR are given in Table 1 .
7
Quantitative miR-200b Expression in Retina
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We performed qRT-PCR for miR-200b expression with retinal samples. miRNA fractions were isolated from retinas using a miRNA isolation kit (CWbio, Co. Ltd.; cat. no. CW0627); U6-rRNA was used as an internal control. The primers sequences for miR-200b and U6-rRNA used for amplification are shown in Table 2 . PCR was carried out using a miRNA Real-time PCR Assay Kit (CWbio Co. Ltd; cat. no. CW214). After the reaction, the Ct values of the samples were calculated at the point at which they reached the threshold value during the process of PCR amplification, and miR-200b levels were quantified based on the ratio of miRNA/U6-rRNA using these 2-ΔΔ Ct method.
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