The expression of miRNAs of interest selected in the TLDA screening was validated using TaqMan miRNA Assays (triplicate per sample) and performed in a Rotor Gene real-time PCR machine (Qiagen, Manchester, UK). After reverse transcription and preamplification with specific TaqMan probes for the miRNAs of interest and endogenous control (snRNAU6), Rotor-Gene 2.1.0.9 software (Qiagen) was used to set the qPCR reaction (initial enzyme activation at 95°C for 10 min, and cycling conditions defined at 95°C for 15 s followed by 60°C for 60 s, for 40 cycles). A no-template control (no PreAmp product) sample was added to test for cDNA contamination.
Taqman mirna assay
Manufactured by Qiagen
Sourced in Germany, United States, China
The TaqMan miRNA assay is a laboratory equipment product designed for the detection and quantification of microRNA (miRNA) molecules. It utilizes real-time PCR technology to measure the expression levels of specific miRNA targets. The assay provides a reliable and sensitive method for analyzing miRNA expression patterns in various biological samples.
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10 protocols using taqman mirna assay
1
Validating miRNA Expression with qPCR
2
Quantitative RT-PCR Analysis of miRNA
3
Quantification of miRNA and mRNA Levels in SW1353 Cells
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Total RNA of SW1353 cells was extracted for analyzing miRNA (Qiagen, U.S.A.) and mRNA (Axygen, U.S.A.) levels according to the manufacturer’s protocols. For quantification of miR-216b, the TaqMan MicroRNA Reverse Transcription Kit and TaqMan miRNA assay (Qiagen, U.S.A.) were used to perform reverse transcription and PCR according to the manufacturer’s instructions. U6 was used as the internal control. The gene expressions of PCNA, Smad3, MMP-13, aggrecan, and type II collagen were detected by using the SYBR Green PCR kits (TAKARA, Japan). β-Actin served as an internal control. The following primers were used: PCNA forward, 5′-CCTGCTGGGATATTAGCTCCA-3′, reverse, 5′-CAGCGGTAGGTGTCGAAGC′; Smad3 forward, 5′-CGGCTCTACTACATCGGAGG-3′, reverse, 5′-GTAGACAGCCTCAAAGCCCT-3′; MMP-13 forward, 5′-TGATGACATCAAGAAGGTGGTGAAG-3′, reverse, 5′-TCCTTGGAGGCCATGTGGGCCAT-3′; aggrecan forward, 5′-TGAGCGGCAGCACTTTGAC-3′, reverse, 5′-TGAGTACAGGAGGCTTGAGG-3′; type II collagen forward, 5′-AGAACTGGTGGAGCAGCAAGA-3′, reverse, 5′-AGCAGGCGTAGGAAGGTCAT-3′; β-actin forward, 5′-AGCGAGCATCCCCCAAAGTT-3′, reverse, 5′-GGGCACGAAGGCTCATCATT-3′; U6 forward, 5′-CGCTTCGGCAGCACATATAC-3′, reverse, 5′-AAATATGGAACGCTTCACGA-3′.
4
Analysis of ErbB3 and miRNA Expression
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Total RNA of tissues and cells was isolated by TRIzol reagent (Invitrogen) following the manufacturer’s protocol. The mRNA expression levels were detected by qRT-PCR. The complementary DNA (cDNA) was obtained by reverse transcription utilizing the miScript II RT Kit (TAKARA, Kusatsu, Japan). The SYBR Green PCR Kits (TAKARA) were employed to conduct quantitative PCR. Primers were synthetized by RiboBio (Guangzhou, P.R. China). The following primers were used: ErbB3, 5′-GGTGATGGGGAACCTTGAGAT-3′ (forward) and 5′-CTGTCACTTCTCGAATCCACTG-3′ (reverse); glyceraldehyde-3-phosphate dehydrogenase (GAPDH), 5′-ACAACTTTGGTATCGTGGAAGG-3′ (forward) and 5′-GCCATCACGCCACAGTTTC-3′ (reverse). Each sample was assessed in triplicate. The expression levels of mRNAs were normalized to levels of GAPDH, which acted as a housekeeping gene.
For the detection of human miRNAs, the TaqMan MicroRNA Reverse Transcription Kit and TaqMan miRNA assay (Qiagen, Hilden, Germany) were used to perform reverse transcription and PCR according to the manufacturer’s instructions. Reverse transcription was performed using the miScript II RT Kit (TAKARA), which was followed by qRT-PCR utilizing the miScript SYBR Green PCR Kit (TAKARA) and hsa-miR-125a-5p and hsa-miR-4319 specific primers that were purchased from RiboBio. The levels of miRNAs were normalized to levels of U6 small nuclear RNA (snRNA).
For the detection of human miRNAs, the TaqMan MicroRNA Reverse Transcription Kit and TaqMan miRNA assay (Qiagen, Hilden, Germany) were used to perform reverse transcription and PCR according to the manufacturer’s instructions. Reverse transcription was performed using the miScript II RT Kit (TAKARA), which was followed by qRT-PCR utilizing the miScript SYBR Green PCR Kit (TAKARA) and hsa-miR-125a-5p and hsa-miR-4319 specific primers that were purchased from RiboBio. The levels of miRNAs were normalized to levels of U6 small nuclear RNA (snRNA).
5
Quantitative Expression Analysis of miR-488 and HMGN5
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Total RNA from tissues or cell lines was isolated by using TRIzol reagent (Thermo Fisher Scientific) according to the manufacturer’s instructions. cDNA was synthesized using PrimeScript RT reagent kit (Takara, Tokyo, Japan). Detection of miR-488 was performed by using a TaqMan miRNA assay in accordance with the manufacturer’s protocols (Qiagen, Shanghai, China) under ABI 7500 Real-time PCR system (Bio-Rad Laboratories Inc., Hercules, CA, USA). U6 was used as the internal control for miRNA. The mRNA expression level of HMGN5 expression was measured by SYBR Green Master Mix (Thermo Fisher Scientific) using ABI 7500 Real-time PCR system (Bio-Rad Laboratories Inc.). Glycer-aldehyde 3-phosphate dehydrogenase (GAPDH) was used as the internal control for mRNA. The relative expression level of miR-488 or HMGN5 was calculated with 2−ΔΔCt method.
6
Quantitative miRNA and mRNA Expression Analysis
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The tissues or cells RNA was isolated with Trizol (Invitrogen, Carlsbad, CA) following the manufacturer’s instructions. The RNA samples were reversed transcribed to complementary DNA using the Prime Script™ RT reagent kit (Takara, Kyoto, Japan) and miRNA expression were performed by TaqMan MicroRNA RT assay and TaqMan MiRNA® Assay (Qiagen, Dusseldorf, Germany), respectively. Quantitative real-time PCR was performed using the SYBR® Premix Ex Taq™ II (Takara, Kyoto, Japan) and the Applied Biosystems 7500 Real-time PCR System (Applied Biosystems, Inc. Carlsbad, CA, USA), according to the manufacturers’ instructions. The mRNA and miRNAs expression were normalized to GAPDH or U6. The primer sequences used in this study were circPVT1 forward primer: 5′-GGTTCCACCAGCGTTATTC-3′, reverse primer: 5′-CAACTTCCTTTGGGTCTCC-3′; GAPDH forward primer: 5′-GTCAACGGATTTGGTCTGTATT-3′ and GAPDH reverse primer: 5′-AGTCTTCTGGGTGGCAGTGAT-3′; MCL-1 forward primer: 5′-GCTTGCTTGTTACACACACAGGTC-3′ and MCL-1 reverse primer: 5′-GCAGAACAATCAGCAATTTCAAGG-3′. Relative mRNA expression was calculated using 2−ΔΔCt methods.
7
Quantifying mRNA and miRNA Expression
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Total RNA was extracted from non-transfected and transfected HK-2 cells using Trizol reagent (Invitrogen, USA), according to the manufacturer's instructions. A First Strand cDNA Synthesis Kit (Invitrogen, USA) was used to generate cDNAs, which were quantified using a DyNamo SYBR1 Green qPCR Kit (Takara, Dalian, China) and gene-specific primers (Table S1–2). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal control. The TaqMan MicroRNA Reverse Transcription Kit and TaqMan miRNA assay (Qiagen, China) were used to quantify miRNA expression; U6 was used as an internal control. Expression levels were calculated using the 2−ΔΔCT method.
8
Analysis of chondrocyte miRNA and gene expression
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Total RNA of chondrocytes was extracted for the analysis of miRNA and mRNA following the manufacturer’s instructions. For quantification of miR-448, the TaqMan MicroRNA Reverse Transcription Kit and TaqMan miRNA assay (Qiagen) were respectively used to perform reverse transcription and PCR following the manufacturer’s instructions. U6 was used as the internal control. The gene expressions of matrilin-3, aggrecan, type II collagen and MMP-13 were detected using the SYBR Green PCR kits (TAKARA). GAPDH served as an internal control. All PCR assays were performed in CFX96 Touch (Bio-Rad). The following primers were used:
Matrilin-3 forward, 5’-TCTCCCGGATAATCGACACTC-3′, reverse, 5’-CAAGGGTGTGATTCGACCCA-3’
Aggrecan forward, 5’-ACCAGACGGGCCTCCCAGAC-3′, reverse, 5’-TGGCTCTGCCCCAGAGGGAC-3′
Type II collagen forward, 5’-TGAGGGCGCGGTAGAGACCC-3′, reverse, 5’-TGCACACAGCTGCCAGCCTC-3’
MMP-13 forward, 5’-ATGCGGGGTTCCTGATGTGG-3′, reverse, 5’-GGCCCAGGAGGAAAAGCATG-3’
GAPDH forward, 5’-ACAACTTTGGTATCGTGGAAGG-3′, reverse, 5’-GCCATCACGCCACAGTTTC-3’
U6 forward, 5’-CGCTTCGGCAGCACATATAC-3′, reverse, 5’-AAATATGGAACGCTTCACGA-3′
9
Analyzing Exosomal MicroRNA Expression
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To explore exosomal microRNAs, miRNA-21 and miRNA-210 were selected [22, 23] . The RNA was reversetranscribed using the TaqMan microRNA Reverse Transcription Kit (Qiagen, Hilden, Germany). The mixture was incubated at 37 °C for 60 min and 94 °C for 5 min. qPCR was performed by using the TaqMan miRNA assay according to the manufacturer's protocol (Qiagen, Hilden, Germany) and Cq values were calculated. The miR-39 value was used as the internal control. The amplification conditions of the mixture were as follows: 95 °C for 15 min, followed by 45 cycles at 94 °C for 15 sec and at 55 °C for 30 sec. The relative miRNA expression values were normalized to Cel-miR-39 and calculated using the the 2 -ΔΔCq method.
10
Profiling miRNA and mRNA Expression
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Total RNA of tissues and HK2 cells was extracted for analyzing miRNA (Qiagen, USA) and mRNA (Axygen, USA) levels according to the manufacturer's protocols. For quantification of miR-23a, the TaqMan MicroRNA Reverse Transcription Kit and TaqMan miRNA assay (Qiagen, USA) were used to perform reverse transcription and PCR according to the manufacturer's instructions. U6 was used as the internal control. The gene expressions of E-cadherin, a-SMA, Vimentin and FN, and Col IV were detected using the SYBR Green PCR kits (TAKARA, Japan). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was amplified as an internal reference. Each sample was assessed in triplicate. Primers used were listed in Table 1.
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