Total RNA was extracted from MKN-45 and MGC-803 cells with Trizol reagent (Takara, Otsu, Japan). A TaqManTM Advanced miRNA cDNA Synthesis Kit (Waltham, MA, United States) or a reverse transcription kit (Takara, Otsu, Japan) was utilized to reverse transcribe the RNAs into cDNA. An quantitative real-time PCR (RT-qPCR) assay was performed with an SYBR Green PCR Kit (Takara, Otsu, Japan). GAPDH and U6 were utilized as internal controls. An Applied Biosystems Step One Plus Real-Time PCR System (Applied Biosystems, Foster City, United States) was used to analyze the results. In addition, the relative expression levels were examined with the 2−ΔΔCt method.
Taqman advanced mirna cdna synthesis kit
Manufactured by Takara Bio
Sourced in United States
The TaqMan™ Advanced miRNA cDNA Synthesis Kit is a tool for converting mature microRNA (miRNA) molecules into cDNA. It enables the synthesis of cDNA from small RNA samples, which can then be used for downstream applications such as real-time PCR analysis.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (5 mentions)
Reverse transcription kit, by Takara Bio (3 mentions)
Sybr green pcr kit, by Takara Bio (3 mentions)
Steponeplus real time pcr system, by Thermo Fisher Scientific (2 mentions)
Trizol reagent, by Takara Bio (2 mentions)
Sybr green mixture, by Takara Bio (1 mentions)
Stepone real time pcr system, by Takara Bio (1 mentions)
Primescript rt kit, by Takara Bio (1 mentions)
Sybr green, by Thermo Fisher Scientific (1 mentions)
Rnase r, by Illumina (1 mentions)
First strand cdna synthesis kit, by Takara Bio (1 mentions)
Abi 7500 fast real time polymerase chain reaction system, by Thermo Fisher Scientific (1 mentions)
Sybr green kit, by Takara Bio (1 mentions)
Abi7500 real time qpcr system, by Thermo Fisher Scientific (1 mentions)
Abi 7500 real time pcr system, by Thermo Fisher Scientific (1 mentions)
Sybr green pcr master mix kit, by Takara Bio (1 mentions)
7 protocols using taqman advanced mirna cdna synthesis kit
1
Quantitative Analysis of miRNA Expression
2
LUAD Cell RNA Extraction and qRT-PCR
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Under the suggestions of manufacturer, total RNA extracted from LUAD cells was isolated with TRIzol reagent (Invitrogen). The isolated RNAs (1 μg) were reverse-transcribed into cDNA by the use of TaqManTM Advanced miRNA cDNA Synthesis Kit (Waltham, MA, USA) or the reverse transcription kit (Takara, Otsu, Japan). qRT-PCR was carried out by employing the StepOneTM Real-Time PCR System and the SYBR® Green Mixture (Takara). GAPDH or U6 was acted as the internal control. The 2−ΔΔCt method was utilized to quantify relative expression levels.
3
Quantifying Circular RNA and Target Gene Expression
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Total RNA was extracted from LUAD tissues and cells with TRIzol reagent (Invitrogen). Reverse transcription was implemented with a PrimeScript RT kit (Takara, Dalian, China) to produce the first-strand complementary DNA (cDNA). miR-6807-3p was reverse transcribed with a TaqMan advanced miRNA cDNA synthesis kit (Takara). Quantitative real-time PCR was performed using SYBR Green (Applied Biosystems, Foster City, CA, USA). circ_0018414, miR-6807-3p, and DKK1 expression levels were counted by the 2−ΔΔCt method, with GAPDH and U6 as the internal controls as needed.
For RNase R treatment, 2 mg of total RNA was incubated with 3 U/mg RNase R (Epicenter Technologies, Madison, WI, USA) for 15 min at 37°C. The group without RNase R treatment served as the mock. The levels of circ_0018414, DKK1, and GAPDH in both groups were evaluated by quantitative real-time PCR.
For RNase R treatment, 2 mg of total RNA was incubated with 3 U/mg RNase R (Epicenter Technologies, Madison, WI, USA) for 15 min at 37°C. The group without RNase R treatment served as the mock. The levels of circ_0018414, DKK1, and GAPDH in both groups were evaluated by quantitative real-time PCR.
4
RNA Extraction and qRT-PCR Analysis
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Total RNA was extracted from PDAC tissues or cells by use of TRIzol Reagent (Invitrogen). Afterwards, RNAs were reversely transcribed into cDNA with TaqMan™ Advanced miRNA cDNA Synthesis Kit (for mRNA and lncRNA; Waltham, MA, USA) or First Strand cDNA Synthesis Kit (for miRNA; Takara, Otsu, Japan). The RT-qPCR analysis was conducted on ABI 7500 Fast Real-Time polymerase chain reaction system (Applied Biosystems, Foster City, CA, USA) via using SYBR Green PCR Kit (Takara, Japan). The relative expression of genes was calculated with the 2−ΔΔCt method. All data were normalized to the expression of U6 or GAPDH.
5
Quantitative Analysis of lncRNA and miRNA Expressions
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Total RNA were extracted from A549 or H1299 cells with the employment of Trizol reagent (Takara, Otsu, Japan). RNAs were reverse transcribed into cDNA by using TaqMan™ Advanced miRNA cDNA Synthesis Kit (Waltham, MA, U.S.A.) or the reverse transcription kit (Takara, Otsu, Japan). The RT-qPCR was conducted by SYBR Green PCR Kit (Takara, Otsu, Japan). Internal controls were GAPDH and U6. The results of RT-qPCR were analyzed by using Applied Biosystems Step One Plus Real-Time PCR System (Applied Biosystems, Foster city, U.S.A.), and the 2−ΔΔCt method was used to examine these relative expression levels. The primers for RT-qPCR were as follows: FBXL19-AS1: 5′-GGT ACA ACT ACG GAT ATG A-3′ (Forward) and 5′-TAC GTC TCG ACC ATT ACG CA-3′ (Reverse); miR-431-5p: 5′-TGT CTT GCA GGC CGT CAT G-3′ (Forward) and 5′-GCT GTC AAC GAT ACG CTA CCT A-3′ (Reverse); RAF1: 5′-GGG AGC TTG GAA GAC GAT CAG-3′ (Forward) and 5′-ACA CGG ATA GTG TTG CTT GTC-3′ (Reverse); GAPDH: 5′-GAA GGT GAA GGT CGG AGT C-3′ (Forward) and 5′-GAA GAT GGT GAT GGG ATT TC-3′ (Reverse); U6: 5′-ATT GGA ACG ATA CAG AGA AGA TT-3′ (Forward) and 5′-GGA ACG CTT CAC GAA TTT G-3′ (Reverse).
6
Quantifying miR-27a and GSK-3β in Breast Cancer
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Total RNA was isolated from breast cancer tissues and cells using TRIzol reagent (Invitrogen). Extracted RNA was reverse transcribed into cDNA using the Taqman Advanced miRNA cDNA Synthesis Kit (Takara, Japan). Based on ABI7500 real-time qPCR system (Thermo Fisher, Waltham, MA, USA), real-time qPCRs were performed using SYBR Green kit (Takara, Dalian, China). The 2−ΔΔCt method was used to calculate the relative expression of miR-27a and GSK-3β. Values were normalized to U6 or GAPDH. Primer sequences are shown in Table 1
7
Quantitative Analysis of Gene Expression
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Total RNA was extracted from NCI-N87 and AGS cells using TRIzol® reagent (Invitrogen; Thermo Fisher Scientific, Inc.). A PrimeScript RT reagent kit (cat. no. RR047A; Takara Biotechnology Co., Ltd.) or a TaqMan™ Advanced miRNA cDNA Synthesis kit (cat. no. 4366596; Thermo Fisher Scientific, Inc.) were used to reverse transcribe RNAs into cDNA according to the manufacturer's protocol. RT-qPCR was performed on the ABI 7500 real-time PCR System (Applied Biosystems; Thermo Fisher Scientific, Inc.) using a SYBR-Green PCR Master Mix kit (Takara Biotechnology Co., Ltd.). The following thermocycling conditions were used: Pre-denaturation at 95°C for 15 sec, denaturation at 94°C for 30 sec, annealing at 60°C for 20 sec, and extension at 72°C for 40 sec for 40 cycles. The expression levels of genes were calculated utilizing the 2−ΔΔCq method (20 (link)), with GAPDH or U6 as the internal controls The primer (Shanghai GeneChem) sequences used in the present study were as follows: MCM3AP-AS1 forward, 5′-CCTATCCCTTTCTCTAAGA-3′, reverse, 5′-ACTTCTGCAAAAACGTGCTG-3′; miR-138 forward, 5′-GTTAGGGCAGGTGGGATG-3′, reverse, 5′-TGTATGCGGCTGGTAAGTAG-3′; FOXC1 forward, 5′-AACATCGCCTGCGTTATCCTC-3′, reverse, 5′-ACGTCCCGGATGATCCCAA-3′; U6 forward, 5′-TTATGGGTCCTAGCCTGAC-3′, reverse, 5′-CACTATTGCGGGTCTGC-3′; and GAPDH forward, 5′-CCACATCGCTCAGACACCAT-3′ and reverse, 5′-ACCAGGCGCCCAATACG-3′.
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