Total RNA from cells was extracted by using the TRIzol reagent (Invitrogen) according to the manufacturer's protocol. For the quantification of miR‐629, cDNA was synthesized by the One Step Prime script miRNA cDNA synthesis kit (Qiagen, Valencia), and real‐time PCR was performed using the miRNA‐specific TaqMan MiRNA Assay Kit (Applied Biosystems, Foster City, USA) on an ABI7900 system (Applied Biosystems). For the mRNA detection, mRNA was reversely transcribed into cDNA using PrimeScript RT reagent kit (Takara, Dalian, China), and real‐time PCR was performed using SYBR Green Premix Ex Taq II kit (Takara) on an ABI7900 system (Applied Biosystems). U6 and GAPDH were used as internal controls for miRNA and mRNA expression, respectively, and the relative expression of respective genes was calculated by 2−ΔΔCt method.
Onestep primescript mirna cdna synthesis kit
Manufactured by Qiagen
Sourced in United States
The OneStep PrimeScript miRNA cDNA Synthesis Kit is a laboratory equipment product designed for the reverse transcription of mature microRNA (miRNA) molecules. The kit enables the conversion of miRNA into complementary DNA (cDNA) in a single-step reaction.
Automatically generated - may contain errors
Lab products found in correlation
Nanodrop 2000 spectrophotometer, by Thermo Fisher Scientific (4 mentions)
Trizol reagent, by Thermo Fisher Scientific (3 mentions)
Primescript rt reagent kit, by Takara Bio (3 mentions)
Sybr premix ex taq 2 kit, by Takara Bio (2 mentions)
Taqman mirna assay kit, by Thermo Fisher Scientific (1 mentions)
Sybr green premix ex taq 2 kit, by Takara Bio (1 mentions)
Abi 7900 system, by Thermo Fisher Scientific (1 mentions)
Reverse transcription kit, by Takara Bio (1 mentions)
Abi 7900 qpcr system, by Thermo Fisher Scientific (1 mentions)
Miscript sybr green pcr kit, by Qiagen (1 mentions)
Sybr green real time pcr master mix, by Roche (1 mentions)
Abi 7500 real time pcr system, by Thermo Fisher Scientific (1 mentions)
Taqman mirna reverse transcription kit, by Thermo Fisher Scientific (1 mentions)
Sybr primescript rt pcr kit, by Takara Bio (1 mentions)
Dual beam ultraviolet spectrophotometer, by Eppendorf (1 mentions)
Lipofectamine 2000 reagent, by Thermo Fisher Scientific (1 mentions)
Mirvana mirna isolation kit, by Thermo Fisher Scientific (1 mentions)
6 protocols using onestep primescript mirna cdna synthesis kit
1
Quantification of miR-629 and mRNA
2
Quantitative Analysis of miRNA and mRNA Expression
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Total RNA was purified from clinical samples and cultured cell lines based on the protocol of TRIzol reagent (Invitrogen). For detection of miR‐133a expression, cDNA was synthesized using One Step Prime script miRNA cDNA Synthesis Kit (Qiagen, Hilden, Germany) following the manufacturer's instructions. Quantitative polymerase chain reaction (qPCR) was conducted using the miScript SYBR Green PCR Kit (Qiagen) under the ABI 7900 qPCR System (Applied Biosystems, Foster, CA). For detection of mRNA expression, cDNAs were synthesized using a reverse transcription kit (Takara, Dalian, China). Quantitative polymerase chain reaction was performed by the SYBR Green Real‐Time PCR Master Mix (Roche, Basel, Switzerland) the manufacturer's instructions. All primes used in this study are listed in Table 2 . Glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH) and U6 small nuclear RNA were used as internal controls. Relative expression levels were calculated based on 2−∆∆Ct method from three independent experiments.
3
Quantitative Analysis of lncRNA and miRNA
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Total RNA was extracted from prepared cell lines or tissues using TRizol reagent (Invitrogen). RNA concentration and quality were measured using a NanoDrop 2000 Spectrophotometer (Thermo Fisher Scientific, Inc., Waltham, MA, U.S.A.). For lncRNA quantitation, RNA was reverse transcribed to cDNA using PrimeScript RT reagent Kit (TaKaRa). For miRNA quantitation, reverse transcription was performed using OneStep PrimeScript miRNA cDNA Synthesis Kit (Qiagen, Valencia, CA, U.S.A.). After reverse transcription, qPCR analysis was performed using SYBR Premix ExTaq II Kit (TaKaRa) on ABI 7500 Real-time PCR System (Life Technologies, Carlsbad, CA, U.S.A.). GAPDH or U6 was used for the normalization of lncRNA and miRNA, respectively. Relative quantitation of tested gene expression was calculated and normalized by the 2−ΔΔCt method [10 (link)]. The sequences of primers used here were listed in Table 2 .
4
Quantifying miR-338-3p and RUNX2 in AML
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Total RNA was extracted from AML cells using TRIzo Reagent (TaKaRa, Dalian, China). Afterwards, cDNA was generated from 1 μg RNA as template by performing reverse transcription PCR. miR-338-3p was quantified by virtue of a TaqMan miRNA Reverse-Transcription Kit (Life Technologies, Carlsbad, CA, USA), and U6 snRNA acted as the internal control. The gene expression of RUNX2 was assessed by using One Step Prime Script miRNA cDNA Synthesis Kit (Qiagen, Valencia, CA, USA) and a SYBR™ PrimeScript RT-PCR Kit (TaKaRa), according to the manufacturer's instructions. Primer sequences were shown in Table 1 . The comparative 2 − ΔΔCt method was used for relative quantification and statistical analysis.
5
Comprehensive Transcriptome Analysis Pipeline
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TRizol reagent was applied for extracting total RNA from prepared tissues or cells. A NanoDrop 2000 spectrophotometer (Thermo, MA, USA) was used for measuring the RNA concentration and quality. PrimeScript RT reagent Kit (TaKaRa) and OneStep PrimeScript miRNA cDNA Synthesis Kit (Qiagen, CA, USA) were used to perform reverse transcription in lncRNA and miRNA quantification, respectively. SYBR Premix Ex Taq II kit (Takara) was used to determine qPCR analysis. GAPDH and U6 were applied for normalization of lncRNA and miRNA, respectively. The expression of tested genes was calculated by 2‐ΔΔCt method. The primers sequences used are listed in Table 1 .
6
Quantifying miR-338-3p in Ovarian Cancer Cells
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Total RNA was extracted from fresh tissues sample and cells (HOSEpiC, SKOV3, OVCAR3 and A2780) using the mirVana miRNA Isolation kit (Ambion, USA), according to the manufacturer's instructions. The purity and concentration of RNA were determined using a dual-beam ultraviolet spectrophotometer (Eppendorf, Hamburg, Germany). Then, the RNA was reversely transcribed into cDNA using One Step Prime script miRNA cDNA Synthesis kit (Qiagen, Valencia, CA, USA) according to the manufacturer's instructions. Then miR-338-3p was quantified as described by Chen et al (18) . U6 snRNA was used as an endogenous control. The comparative 2 -∆∆Ct method was used for relative quantification and statistical analysis.
Transfection of cells with miR-338-3p. The miR-338-3p mimic or corresponding negative control (miR-NC) were purchased from Shanghai GenePharma (Shanghai, China). To transfect cells, 50 nM of miR-338-3p or miR-NC was diluted in 500 µl of serum-free media and 5 µl of Lipofectamine 2000 reagent (Invitrogen, Grand Island, NY, USA) according to the manufacturer's instructions. The transfection mixture was added to 1x10 6 cells in a 60-mm dish containing 4 ml medium supplemented with 10% FbS. The cells were harvested 24, 48 and 72 h after transfection and prepared for the subsequent study. Transfection efficiencies were evaluated in every experiment by qRT-PCR 48-h post-transfection.
Transfection of cells with miR-338-3p. The miR-338-3p mimic or corresponding negative control (miR-NC) were purchased from Shanghai GenePharma (Shanghai, China). To transfect cells, 50 nM of miR-338-3p or miR-NC was diluted in 500 µl of serum-free media and 5 µl of Lipofectamine 2000 reagent (Invitrogen, Grand Island, NY, USA) according to the manufacturer's instructions. The transfection mixture was added to 1x10 6 cells in a 60-mm dish containing 4 ml medium supplemented with 10% FbS. The cells were harvested 24, 48 and 72 h after transfection and prepared for the subsequent study. Transfection efficiencies were evaluated in every experiment by qRT-PCR 48-h post-transfection.
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