Total RNA was isolated from BCA samples and cells using TRIzol reagent (Invitrogen, Carlsbad, CA, United States). Sample RNA (1 μg) was reverse transcribed using a Reverse Transcription Kit (Takara, Dalian, China). Quantitative real-time PCR was performed using an SYBR Green Real-time PCR Master Mix kit (Toyobo, Osaka, Japan) on the 7900HT fast real-time PCR system (Applied Biosystems, San Francisco, CA, United States). The levels of miR-99b were quantified with the mirVanaTM qRT-PCR microRNA Detection Kit (Ambion, Austin, TX, United States). The primers used in this study were synthesized by Genepharma (Shanghai, China). The primer sequences for qRT-PCR were purchase from Genepharma (Shanghai, China). The results were normalized to the levels of GAPDH or U6, respectively.
Mirvanatm qrt pcr microrna detection kit
Manufactured by Thermo Fisher Scientific
Sourced in United States, Japan
The MirVanaTM qRT-PCR microRNA Detection Kit is a laboratory product designed for the detection and quantification of microRNA (miRNA) molecules using quantitative reverse transcription polymerase chain reaction (qRT-PCR) technology.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (8 mentions)
Sybr green quantitative real time pcr master mix kit, by Toyobo (5 mentions)
M mlv reverse transcriptase kit, by Thermo Fisher Scientific (3 mentions)
Biosystems 7300 real time pcr system, by Thermo Fisher Scientific (2 mentions)
Quantitect reverse transcription kit, by Qiagen (2 mentions)
Rneasy mini kit, by Qiagen (2 mentions)
Prism 4, by GraphPad (2 mentions)
Sybr green rt pcr kit, by Takara Bio (2 mentions)
Ne per nuclear and cytoplasmic extraction reagent, by Thermo Fisher Scientific (2 mentions)
Reverse transcription kit, by Takara Bio (1 mentions)
Sybr green real time pcr master mix kit, by Toyobo (1 mentions)
7900ht fast real time pcr system, by Thermo Fisher Scientific (1 mentions)
Trizol, by Thermo Fisher Scientific (1 mentions)
Primescript rt reagent kit, by Takara Bio (1 mentions)
Primescript 2 1st strand cdna synthesis kit, by Takara Bio (1 mentions)
Abi 7500 rt pcr system, by Thermo Fisher Scientific (1 mentions)
Gapdh, by GenePharma (1 mentions)
Sybr pre mix ex taq quantitative pcr kit, by Takara Bio (1 mentions)
Primescript rt master mix, by Takara Bio (1 mentions)
Rnase r, by Geneseed (1 mentions)
13 protocols using mirvanatm qrt pcr microrna detection kit
1
Quantification of miR-99b Expression
2
Quantitative Analysis of miR-26a/26b and ST Genes
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Total RNA was extracted from frozen tissues and breast cancer cell lines, using the RNeasy Mini Kit (Qiagen, valencia, CA, USA), and the purity of the preparation was checked by ratio of the absorbance at 260 and 280 nm. The cDNA was synthesized with 2 μg of RNA using QuantiTect Reverse Transcription Kit (Qiagen). The expression of miR-26a/26b was determined by using mirVanaTM qRT-PCR microRNA Detection Kit (Ambion Inc., Austin, TX, USA) and normalized using U6 snRNA and RNU48 as control. ST mRNA was quantified with SYBR Green Quantitative Real-Time PCR Master Mix Kit (Toyobo Co., Osaka, Japan) and normalized to GAPDH and β-action, respectively. The expression level of ST target genes was determined by using Biosystems 7300 Real-Time PCR System (ABI, Foster City, CA, USA). The sequences of primers were as shown in Table 2 . Three independent experiments were each performed in triplicate.
3
Quantification of miRNAs and CTGF mRNA
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Trizol reagent (Invitrogen, Carlsbad, CA) were used to extract total RNA in accordance with the manufacturer’s protocol. Quantitative real-time PCR was performed with mirVanaTM qRT-PCR microRNA detection kit (Ambion, Austin, TX) to determine the relative expression level of miRNAs (miR-574-3p, -543-5p, 455-3p, 455-5p, and 199a-5p) in accordance with the manufacturer’s protocol. Real-time RT-PCR was performed with the standard SYBR Green RT-PCR Kit (Takara, Otsu, Japan) to detect the expression of CTGF mRNA in accordance with the manufacturer’s protocol. The primer set for determination of CTGF mRNA expression level was: forward, 5′-ACAAGGGCCTCTTCTGTGACTT-3′ and reverse, GGTACACCGTACCACCGAAGAT-3′. 2-DDCt method and the GraphPad Prism 4.0 software (GraphPad Software, San Diego, CA) were used to qualify the relative expression of miRNAs or CTGF mRNA and the miRNAs with U6 as an internal control. 2−DDCt method was used to calculate the relative expression level of the miRNAs and the mRNA.
4
Subcellular RNA Extraction and qPCR Analysis
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Total RNAs were extracted from tissues and cells by employing TRIzol (Invitrogen) following the manufacturer’s instructions. To obtain nuclear and cytosolic RNAs, a PARIS Kit was used to separate subcellular fractions of cells before extracting RNAs. RNAs were reversely transcribed into cDNAs with a PrimeScript RT reagent kit (Takara, Japan). qPCR was carried out using SYBR real-time PCR kits (Takara). mRNA expression was calculated based on an internal control GAPDH. Expression of miRNAs were detected by a mirVanaTM qRT-PCR microRNA Detection Kit (Ambion, USA) and normalized to U6 expression.
5
Quantifying miRNA and mRNA Expressions
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Trizol reagent (Invitrogen, CA, US) was used to extract total RNA from HL-1 and 293 cells or tissue samples. RNeasy Qiagen columns (Qiagen Inc., Valencia, CA) were used to purify the extracted RNA, which was then reversely transcribed into cDNA using a PrimeScript II 1st Strand cDNA Synthesis Kit (Takara Bio Inc., Japan) following a standard protocol. The relative expression of miR-125a was measured using qRT-PCR and a mirVanaTM qRT-PCR microRNA detection kit (Ambion, Austin, TX), while the relative expression of IL-6R and IL-16 mRNA was measured using qRT-PCR and a standard SYBR Green RT-PCR Kit (Takara, Otsu, Japan). The expression of miR-125a, IL-6R mRNA, and IL-16 mRNA was quantified using the 2−ΔΔCt method and GraphPad Prism 4.0 software (GraphPad Software, San Diego, CA). Each experiment was run in triplicate.
6
Quantitative Analysis of lncRNA and miRNA
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Total RNA was extracted from tissues or cells using TRIzol reagent (Invitrogen, Carlsbad, CA, United States), and cDNA was synthesized from 1 μg of RNA with an M-MLV Reverse Transcriptase Kit (Invitrogen, Carlsbad, CA, United States). The qRT-PCR analysis was performed using the SYBR Green quantitative real-time PCR Master Mix kit (Toyobo, Osaka, Japan) on an ABI-7500 RT-PCR system (Applied Biosystems). The primers for LINC00319, RAP2A and GAPDH were obtained from GenePharma (Shanghai, China). For the measurement of miR-3127 expression, we used the mirVanaTM qRT-PCR microRNA Detection Kit (Ambion, Austin, TX, United States) according to the manufacturer’s instructions. The relative expression of miR-3127 was normalized against that of the U6 endogenous control.
7
Circular RNA and Gene Expression Analysis
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Total RNA from cells and freshly frozen tissues was isolated using TRIzol reagent (Invitrogen). The total RNA was reverse transcribed using the PrimeScript RT Master Mix (Takara, Dalian, China). Total RNA was incubated at 37°C with 3 U/μg RNase R (Geneseed, Guangzhou, China) for 30 min. Nuclear and cytoplasmic RNA fractionation was isolated using NE-PER Nuclear and Cytoplasmic Extraction Reagents (Thermo Fisher Scientific, CA. United States). The expression of circ_0001666 and SOX4 was quantified using SYBR Pre-mix Ex Taq quantitative PCR kit (Takara, Dalian, China). GAPDH was used as an internal control for circ_0001666 and SOX4. The expression of miR-1251 was determined using the mirVanaTM qRT-PCR microRNA Detection Kit (Ambion, Austin, TX, United States) and normalized to U6. The primer sequences were obtained from Ribobio (Guangzhou, China).
8
Quantitative Analysis of miRNA and FUT mRNA Expression
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Total RNA was isolated from frozen tissues and HCC cell lines, using the RNeasy Mini Kit (QIAGEN, valencia, CA), and cDNA was synthesized using QuantiTect Reverse Transcription Kit (QIAGEN, valencia, CA) according to the manufacturer's protocol. The expression of miRNAs was determined by using mirVanaTM qRT-PCR microRNA Detection Kit (Ambion Inc., Austin, TX, USA) and normalized using the 2-ΔΔCT method relative to U6-small nuclear RNA. FUT mRNAs were quantified with SYBR-Green-quantitative real-time PCR Master Mix kit (Toyobo Co., Osaka, Japan) and normalized to GAPDH, respectively. The expression level of target genes was determined by using Biosystems 7300 Real-Time PCR system (ABI, Foster City, CA, USA) and calculated as 2−(CtTarget gene− CtGAPDH). All assays were triplicated independently. The sequences of primers were as shown Table 3 .
9
Quantifying Gene and miRNA Expression
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According to the manufacturer’s protocol, total RNAs were isolated from BEAS-2B cells with Trizol kit (Invitrogen). Two micrograms of RNA was transcribed into cDNA, and the samples were subjected to qRT-PCR analysis with an SYBR Premix Ex TaqTM kit (Takara, Tokyo, Japan) or mirVanaTM qRT-PCR microRNA Detection kit (Ambion, TX, USA) [11–13 (link)]. The primers of PCR are listed in Supplemental Table S1. GAPDH and U6 were employed as internal references for SOX2-OT/phosphatase and tensin homolog (PTEN) and miR-455-3p. Target gene expression was quantified by the 2−ΔΔCt method.
10
Quantitative Analysis of DIO3OS and miR-122
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Total RNA was isolated from cells using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) and then was converted to cDNA using an M-MLV Reverse Transcriptase Kit (Invitrogen, Carlsbad, CA, USA). Real-time PCR analysis was carried out using the SYBR-Green-quantitative real-time PCR Master Mix kit (Toyobo, Osaka, Japan). The primers for DIO3OS and GAPDH have been reported [5 (link)]. GAPDH served as the endogenous control. For detecting miRNA expression, the mirVanaTM qRT-PCR microRNA Detection Kit (Ambion Inc., Austin, TX, USA) was used according to the manufacturer’s instructions. MiR-122 expression was normalized to U6.
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