RNA was extracted from frozen lungs of 3-week-old mice using TRIzol reagent (Life Technologies) and quantified by Nanodrop spectroscopy (Thermo Scientific). cDNA was generated from 1 μg RNA by reverse transcription using qScript cDNA synthesis kit (Quanta Biosciences, Gaithersburg, MD). Real-time quantitative polymerase chain reaction was performed on a StepOne PCR system (Applied Biosystems, Foster City, CA) using TaqMan probes (Life Technologies) for GSNOR (Mm00475804_g1) compared with 25% diluted β-actin control (Thermo Scientific) with PerfeCTa qPCR FastMix, UNG, ROX (Quanta Biosciences, Gaithersburg, MD). For microRNA qRT-PCR, RNA was similarly extracted as in the miR microarray studies. cDNA was generated using TaqMan primer-specific assays and MicroRNA Reverse Transcription kit, and real-time quantitative polymerase chain reaction was performed using TaqMan MicroRNA assays for microRNA-342-3p (2260, Thermo Scientific) compared with snRNA-U6 control (001973, Thermo Scientific) with TaqMan Universal Master Mix, No AmpErase UNG (Life Technologies). Fold changes are reported utilizing 2^-ddCT method and StepOne software v2.3 (Applied Biosystems).
Microrna reverse transcription kit
Manufactured by Thermo Fisher Scientific
Sourced in United States, China, Japan
The MicroRNA Reverse Transcription Kit is a laboratory tool designed to convert mature microRNA (miRNA) molecules into complementary DNA (cDNA) for subsequent analysis. The kit provides the necessary reagents and protocols to efficiently perform reverse transcription of miRNA samples.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (38 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (13 mentions)
Taqman microrna assay, by Thermo Fisher Scientific (10 mentions)
Trizol, by Thermo Fisher Scientific (9 mentions)
Primescript rt reagent kit, by Takara Bio (8 mentions)
Rneasy mini kit, by Qiagen (5 mentions)
Taqman universal pcr master mix, by Thermo Fisher Scientific (5 mentions)
Mirneasy kit, by Qiagen (4 mentions)
Revertaid first strand cdna synthesis kit, by Thermo Fisher Scientific (4 mentions)
Mirneasy mini kit, by Qiagen (4 mentions)
Abi 7500ht system, by Thermo Fisher Scientific (3 mentions)
Light cycler instrument, by Bio-Rad (3 mentions)
7300 real time pcr system, by Thermo Fisher Scientific (3 mentions)
Cdna reverse transcription kit, by Thermo Fisher Scientific (3 mentions)
Taqman microrna assay kit, by Thermo Fisher Scientific (3 mentions)
Abi 7500 real time pcr system, by Thermo Fisher Scientific (3 mentions)
Taqman mirna assay, by Thermo Fisher Scientific (3 mentions)
Nanodrop nd 1000, by Thermo Fisher Scientific (2 mentions)
Sybr green pcr kit, by Takara Bio (2 mentions)
Transzol, by Transgene (2 mentions)
97 protocols using microrna reverse transcription kit
1
Quantitative Gene/miRNA Expression in Mouse Lungs
2
Quantitative Analysis of Circulating miRNAs
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qRT-PCR was performed using a StepOnePlus Real-Time PCR System (Applied Biosystems, Foster City, CA, USA). Pre and postoperative OCC and AEH blood samples were used. The RNA concentration was adjusted to 2.0 ng/μL. The miRNAs were reverse transcribed using a microRNA reverse transcription kit (Thermo Fisher Scientific Inc.) and qPCR was performed according to the manufacturer’s instructions (Thermo Fisher Scientific Inc.). The following primers were used for PCR: miR−21−5p, miR−30a−5p, miR−30c−5p, miR−141−3p, miR−182−5p, miR−183−5p, miR−200a−5p, miR−200a−3p, miR−200b−5p, miR−200b−3p, miR−200c−3p, miR−210−3p, miR−652−3p, and RNU48 (#4427975; Thermo Fisher Scientific Inc.). RNU48 was used as the normalization control. qPCR of miRNA was performed three times in triplicate wells. The relative expression of miRNAs was calculated using the 2−ΔΔCq method.
3
Quantification of miR-338-3p and WNT2B Expression
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Total RNA from cells and tissues was extracted with TRIzol reagent (Thermo Fisher Scientific). The quality of samples was regard as good when OD260 nm/OD280 nm values were greater than 1.8. The Prime Script RT Reagent kit (Takara, Dalian, China) and microRNA Reverse Transcription kit (Thermo Fisher Scientific) were employed to reverse transcribe RNA into complementary DNA. The expression levels of miR-338-3p and WNT2B were analyzed using SYBR Premix Ex Taq II (Takara) for real-time quantitative polymerase chain reaction (RT-qPCR) on an ABI 7500 HT system (Applied Biosystems, Foster City, CA, USA). Relatively quantitative analysis was performed for mean values using the 2−ΔΔCt method. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) or endogenous small nuclear RNA U6 served as an internal control.
The following primers were utilized: miR-338-3p (forward, 5′-ATCCAGTGCGTGTCGTGG-3′; reverse, 5′-TGCTTCCAGC ATCAGTGAT-3′); WNT2B (forward, 5′-ATTTCCCGCTCTGG AGATTT-3′; reverse, 5′-AAGCTGGTGCAAAGGAAAGA-3′); GAPDH (forward, 5′-TCCCATCACCATCTTCCAGG-3′; reverse, 5′-GATGACCCTTTTGGCTCCC-3′); and U6 (forward, 5′-CTCGCTTCGGCAGCACA-3′; reverse, 5′-AACGCTTCACG AATTTGCGT-3′).
The following primers were utilized: miR-338-3p (forward, 5′-ATCCAGTGCGTGTCGTGG-3′; reverse, 5′-TGCTTCCAGC ATCAGTGAT-3′); WNT2B (forward, 5′-ATTTCCCGCTCTGG AGATTT-3′; reverse, 5′-AAGCTGGTGCAAAGGAAAGA-3′); GAPDH (forward, 5′-TCCCATCACCATCTTCCAGG-3′; reverse, 5′-GATGACCCTTTTGGCTCCC-3′); and U6 (forward, 5′-CTCGCTTCGGCAGCACA-3′; reverse, 5′-AACGCTTCACG AATTTGCGT-3′).
4
Quantifying miRNA Expression Profiles
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Approximately, 100 ng of RNA was reverse transcribed using the MicroRNA Reverse Transcription Kit (ThermoFisher Scientific, Australia) with miRNA‐specific TaqMan assays for hsa‐miR‐1301‐3p, hsa‐miR‐769‐5p, hsa‐miR‐423‐5p, hsa‐miR‐451a, hsa‐miR‐493‐3p, hsa‐miR‐370‐3p, and U6 snRNA (Assay ID: 002827, 001998, 002340, 001141, 002364, 002275 and 001973, respectively). Experimental procedures and thermos‐cycling conditions were described previously (Denham et al. 2017a ). Relative miRNA abundance was calculated using the 2‐delta‐delta Ct method and expressed as fold change. All intra‐assay coefficient of variations for miRNAs were, on average, <1%.
5
Quantifying miR-200b Expression
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Total RNA was extracted using TRIzol reagent (Thermo Fisher Scientific) according to the manufacturer’s instructions. MicroRNA reverse transcription kit (Thermo Fisher Scientific) was used to convert 10 ng of total RNA into complementary DNA, according to the manufacturer’s instructions. The miRNA expression was determined on ABI 7500 thermocycler (Thermo Fisher Scientific) using PrimeScript® miRNA RT-PCR Kit (Takara, Dalian, People’s Republic of China), in accordance with the manufacturer’s instructions. The polymerase chain reaction (PCR) conditions were 95°C for 10 minutes followed by 40 cycles of denaturation at 95°C for 15 seconds and annealing/elongation step at 60°C for 1 minute. The relative miR-200b expression was normalized to U6. The relative expression was analyzed by the 2−ΔΔCt method.15 (link)
6
Quantitative Analysis of miRNA and Gene Expression
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Total RNA was reverse transcribed with microRNA Reverse Transcription Kit (Thermo Fisher Scientific) and expression analysed with TaqMan® assay and probes for hsa-miR-21-5p (Thermo Fisher Scientific) and RNU48 as per the manufacturer’s instructions. For gene expression, High-Capacity RNA-to-cDNA™ Kit (Thermo Fisher Scientific) and SYBR™ Green qPCR with SYBR™ Select Master Mix (Thermo Fisher Scientific) were used. Specific primers were designed for each gene transcript by Primer3 (http://bioinfo.ut.ee/primer3-0.4.0/ ) and are listed in Online Appendix: Reagents. All reactions, including no-template controls, were run in Bio-Rad C-1000 touch thermocycler (Bio-Rad, Hercules, CA, USA) and Applied Biosystems® StepOnePlus™ Real-Time PCR system (Thermo Fisher Scientific). Each sample was tested in triplicate. Analysis of data was performed with SDS 2.4.1 software (Thermo Fisher Scientific). Relative changes in expression were evaluated by using the 2ΔCt formula.
7
Quantifying lncRNA and miRNA Levels in Breast Cancer
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Total RNA from BC tissues, noncancerous tissues, and BC cells was extracted using TRIzol® reagent in accordance with the manufacturer's instructions, which were reversely transcribed using TaqMan™ mRNA or microRNA Reverse Transcription Kit (ThermoFisher Scientific). Based on primers generated by Sangon (Shanghai, China),RT-PCR was performed using SYBR Premix Ex TaqI. HOTAIR (Forward, 5′-GGTAGAAAAAGCAACCACGA AGC-3′; Reverse, 5′- ACATAAACCTCTGTCTGTGAGTGCC-3′); miR-130a-3p (Forward: 5′-GATGCTCTCAGTGCAATGTTA-3′;Reverse:5′-CTCTGTCTCTCGTCTTGTTGGT AT-3′).GAPDH(FW:5′-AATGGGCAGCCGTTAGGAAA-3′,RV:5′-GCCCAATACGACCAAATCAGAG-3′),U6(FW:5ʹ-CTCGCTTCGGCAGCACA-3ʹ; RV: 5ʹ-AACGCTTCACGAATTT GCGT- 3ʹ. All samples were prepared in triplicates. The 2-ΔΔCt method was used to calculate the relative RNA expression.
8
Quantification of miR-1307-3p Expression
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Total RNA was extracted form tissues and cell lines using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.). cDNA was synthesized using a MicroRNA Reverse Transcription kit (Thermo Fisher Scientific, Inc.) according to the manufacturers' instructions. The relative miR-1307-3p expression level was determined by RT-qPCR on the 7300 Real-Time PCR System (Applied Biosystems; Thermo Fisher Scientific, Inc.) using the SYBR Green I Master Mix kit (Invitrogen; Thermo Fisher Scientific, Inc.). The reaction were performed as follows: 95°C for 2 min, 40 cycles of 94°C for 30 sec, and 60°C for 30 sec. The relative expression level of miR-1307-3p was normalized to endogenous control U6 and was expressed as 2−∆∆Cq (21 (link)). The sequences of the primers were as follows: miR-1307-3p, forward 5′-TCGGCAGGACTCGGCGTGGCGT-3′, reverse 5′-CTCAACTGGTGTCGTGGA-3′; and U6, forward 5′-CTCGCTTCGGCAGCACA-3′ and reverse 5′-AACGCTTCACGAATTTGCGT-3′.
9
Real-Time qPCR Analysis of Gene Expression
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Tissue and cells (seeded into a 6-well plate at a density of 4x104 cells/well and cultured for 24 h) were collected to extract total RNA using TRIzol® reagent (Thermo Fisher Scientific, Inc.). RNA was reversely transcribed into cDNA using the cDNA Reverse Transcription kit (Thermo Fisher Scientific, Inc.) or MicroRNA Reverse Transcription kit (Thermo Fisher Scientific, Inc.) according to the manufacturer's protocols. The expression levels were determined using the SYBR PremixEx Taq II kit (Takara Biotechnology Co., Ltd.) with GAPDH as an internal control on ABI 7500 RT-PCR system (Applied Biosystems; Thermo Fisher Scientific, Inc.). The qPCR thermocycling conditions were: Initial denaturation at 95˚C for 10 min; followed by 40 cycles of 95˚C for 15 sec and 64˚C for 30 sec. The relative gene expression was analyzed by using the 2-ΔΔCq method (11 (link)). The experiment was repeated three times. The primer sequences were: SNHG16: forward: 5'-GCAGAATGCCATGGTTTCCC-3'; SNHG16: reverse: 5'-GGACAGCTGGCAAGAGACTT-3'; miR-183: forward: 5'-CGCGGTATGGCACTGGTAGA-3'; miR-183: reverse: 5'-AGTGCAGGGTCCGAGGTATTC-3'; FOXO1: forward: 5'-GGATGGCATGTTCATTGAGCG-3'; FOXO1: reverse: 5'-ACTGCTTCTCTCAGTTCCTGC-3'; GAPDH: forward: 5'-CATGAGAAGTATGACAACAGCCT-3'; GAPDH: reverse: 5'-AGTCCTTCCACGATACCAAAGT-3'; U6: forward: 5'-CTCGCTTCGGCAGCACA-3'; U6: reverse: 5'-AACGCTTCACGAATTTGCGT-3'.
10
Dental Pulp Cell RNA Extraction
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Total RNA in the hDPCs and rat incisor pulp tissues was extracted using QuickGene RNA cultured cell kit S (FUJIFILM Wako Pure Chemical) and TRIzol reagent (Thermo Fisher Scientific), respectively. cDNA was synthesized using PrimeScript™ RT Master Mix (Takara Bio, Kusatsu, Japan), and PCR products were amplified using synthesized cDNA, specific primers (Table 1 ), and GoTaq qPCR Master Mix (Promega, Madison, WI, USA) via the CFX96 Real-Time qPCR System (Bio-Rad, Kidlington, UK). Actin beta (ACTB) was used as an internal control. To evaluate miRNA, mirVana miRNA Isolation Kit (Thermo Fisher Scientific) was used to extract total RNA. cDNA was synthesized using TaqMan microRNA Assays (Thermo Fisher Scientific) and a microRNA Reverse Transcription Kit (Thermo Fisher Scientific) with specific RT primers for hsa-miR-146b-5p, rno-miR-146b-5p, and U6. Real-time qPCR was performed with TaqMan Universal Master Mix II, via a CFX96 Real-Time qPCR System (Bio-Rad). U6 spliceosome RNA was used as an internal control.
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