Total RNA from the cultured cells, with the efficient recovery of small RNAs, was isolated using the mirVana miRNA isolation kit (Ambion, Austin, TX, USA). The detection of the mature form of miRNAs was performed using the mirVana qRT-PCR miRNA detection kit and qRT-PCR Primer Sets, according to the manufacturer's instructions (Ambion). The U6 small nuclear RNA was used as an internal control.
Qrt pcr primer sets
Manufactured by Thermo Fisher Scientific
Sourced in United States
QRT-PCR Primer Sets are designed for quantitative real-time PCR (qRT-PCR) experiments. They provide specific primer sequences for the amplification of target gene sequences. These primer sets are optimized for sensitive and accurate quantification of gene expression levels.
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Lab products found in correlation
Mirvana qrt pcr mirna detection kit, by Thermo Fisher Scientific (7 mentions)
Mirvana mirna isolation kit, by Thermo Fisher Scientific (5 mentions)
Rneasy kit, by Qiagen (3 mentions)
Sybr green pcr kit, by Thermo Fisher Scientific (2 mentions)
Mirvana mir isolation kit, by Thermo Fisher Scientific (1 mentions)
Abi 7500 fast real time pcr system, by Thermo Fisher Scientific (1 mentions)
Rt premix, by Bioneer (1 mentions)
Taqman gene expression assay kit, by Thermo Fisher Scientific (1 mentions)
Trizol, by Thermo Fisher Scientific (1 mentions)
Taqman universal master mix 2, by Thermo Fisher Scientific (1 mentions)
Superscript 2 reverse transcriptase kit, by Thermo Fisher Scientific (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
7500 real time pcr system, by Thermo Fisher Scientific (1 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (1 mentions)
10 protocols using qrt pcr primer sets
1
Efficient Extraction and Quantification of miRNAs
2
Quantitative Analysis of miRNA and mRNA
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RNeasy kits (Qiagen, Valencia, CA, USA) were performed to extract total mRNA from cells. miRNAs was extracted by mirVANA miRNA isolation kit (Ambion, Austin, TX, USA). The mirVana qRT-PCR miRNA Detection Kit (Ambion, Austin, TX, USA) and qRT-PCR Primer Sets were utilized to detect the expression of miR-210. qRT-PCR (quantitative real-time PCR) was performed on ABI 7500 with SYBR green PCR kits (Applied Biosystems). Data were normalized by using 2−ΔΔCt method as relative quantification. The results of miRNAs or mRNA by qRT-PCR were normalized to U6 RNA or GAPDH expression level, respectively. The primers were used as follows. HIF-1α: F-ATC GCG GGG ACC GAT T and R-CGA CGT TCA GAA CTT ATC TTT TTC TT. PHD-2: F-ACC ATG AAC AAG CAC GGC ATC TGC and R-GAC GTC TTT GCT GACTGA ATT GGG CTT. BCL-xL: F-CTG TGC GTG GAA AGC GTA G and R-CTC GGC TGC TGC ATT GTT C. GAPDH: F-GCA CCG TCA AGG CTG AGA AC and R-ATG GTG GTG AAG ACG CCA GT.
3
Quantification of miR-485-5p Expression
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Total RNA from the cultured cells, with efficient recovery of small RNAs, was isolated using the mirVana miRNA isolation kit (Ambion, Austin, TX, USA). Detection of the mature form of miRNAs was performed using the mirVana qRT-PCR miRNA detection kit and qRT-PCR primer sets, according to the manufacturer's instructions (Ambion). The sequences of the primers were as follows: miR-485-5p forward, 5′-CCAAGCTTCACCCATTCCTAACAGGAC-3′ and reverse, 5′-CGGGATCCGTAGGTCAGTTACATGCATC-3′. The U6 small nuclear RNA was used as an internal control.
4
Efficient miRNA Isolation and Detection
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Total RNA from cultured cells, with efficient recovery of small RNAs, was isolated using the mirVana miR Isolation kit (Ambion, Thermo Fisher Scientific, Inc.). Detection of the mature form of miRNAs was performed using the mirVana qRT-PCR miR detection kit and qRT-PCR primer sets, according to the manufacturer's instructions (Ambion, Thermo Fisher Scientific, Inc.). The U6 small nuclear RNA was used as an internal control.
5
Quantifying miR-200a Expression
6
Quantification of miR-200a Expression
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The abundance of miR-200a was determined by microRNA real-time PCR assay using the mirVana qRT–PCR miRNA Detection Kit (Thermo Fisher Scientific) and QRT–PCR Primer Sets, according to the manufacturer's instructions (Ambion Inc., Austin, TX, USA). The expression of U6-snRNA was used as an internal control.
7
Quantifying Cellular Gene and miRNA Levels
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Total RNA was extracted from cells with RNeasy kit (Qiagen, Valencia, CA, USA). qRT-PCR was conducted on ABI 7500 with SYBR green PCR kits (Applied Biosystems). GAPDH gene was named as endogenous control. Cellular miRNA was isolated using the mirVana miRNA Isolation Kit (Ambion, Austin, TX, USA). MirVana qRT-PCR miRNA Detection Kit (Ambion) and qRT-PCR Primer Sets (Ambion) was used. The U6 small nuclear RNA was internal control of miRNAs. The data were normalized by 2 -∆∆Ct method as relative quantification. The primers used were as follows. ATM (GenBank accession number NM_000051.3): F:5'-TGG GCT CTG GAA TCA TAC GGC-3', R: 5'-TAA CGC TCA CGA GTG CTC ACC AC-3'. Chk1 (GenBank accession number AF016582.1):F: 5'-AAC GCC TTC CTT GAT GGA AT -3' and R:5'-GCGACTTGAACG-GAATATCTGT -3'. H2AX (GenBank accession number NM_002105.2):F: 5'-CCC TCG GGC GGC AAG AA -3' and R: 5'-GGA GGG CGG ACG GCG GAC AGG -3'. GAPDH: F:5'-TTT TCC CTC TTC TTG ACT CAC CC-3' and R: 5'-GTG CCT TTC ATT CCA TCC AGC-3'.
8
Bioinformatic Analysis of miRNA Targets
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The analysis of potential microRNA target site using the commonly used prediction algorithms -miRanda (http://www.microrna.org/).
Real-time PCR for microRNAs. Total RNA from cultured cells, with efficient recovery of small RNAs, was isolated using the mirVana miRNA Isolation kit (Ambion). Detection of the mature form of miRNAs was performed using the mirVana qRT-PCR miRNA Detection kit and qRT-PCR Primer Sets, according to the manufacturer's instructions (Ambion). The U6 small nuclear RNA was used as an internal control.
Reverse transcription-polymerase chain reaction. It was performed as described before (31) . Primers for VSIG4: forward, 5'-GTGTCCAGTTTGGCTAGTGCC-3'; reverse, 5'-GACTGGAGAACAGAAGCAGGC-3'. Primers for GAPDH: forward, 5'-CGGAGTCAACGGATTTGGTCG TAT-3'; reverse, 5'-AGCCTTCTCCATGGTGGTGAAGAC-3'.
Northern blot analysis. Northern blot analysis for miRNAs were performed as described previously (32) . Probes were labeled with [γ-32 P]-ATP complementary to let-7g-5p and U6 snRNA.
Statistical analysis. Data are presented as mean ± SEM. Student's t-test (two-tailed) was used to compare two groups (P<0.05 was considered significant), unless otherwise indicated (χ 2 test).
Real-time PCR for microRNAs. Total RNA from cultured cells, with efficient recovery of small RNAs, was isolated using the mirVana miRNA Isolation kit (Ambion). Detection of the mature form of miRNAs was performed using the mirVana qRT-PCR miRNA Detection kit and qRT-PCR Primer Sets, according to the manufacturer's instructions (Ambion). The U6 small nuclear RNA was used as an internal control.
Reverse transcription-polymerase chain reaction. It was performed as described before (31) . Primers for VSIG4: forward, 5'-GTGTCCAGTTTGGCTAGTGCC-3'; reverse, 5'-GACTGGAGAACAGAAGCAGGC-3'. Primers for GAPDH: forward, 5'-CGGAGTCAACGGATTTGGTCG TAT-3'; reverse, 5'-AGCCTTCTCCATGGTGGTGAAGAC-3'.
Northern blot analysis. Northern blot analysis for miRNAs were performed as described previously (32) . Probes were labeled with [γ-32 P]-ATP complementary to let-7g-5p and U6 snRNA.
Statistical analysis. Data are presented as mean ± SEM. Student's t-test (two-tailed) was used to compare two groups (P<0.05 was considered significant), unless otherwise indicated (χ 2 test).
9
Quantitative Expression Analysis of Pigmentation Genes
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To determine relative mRNA expression of selected genes, total RNA was isolated with TRIzol (Invitrogen, CA, USA), according to the manufacturer’s instructions, and 4 µg RNA was reverse-transcribed into cDNA using RT-premix (Bioneer, Seoul, South Korea). Quantitative PCR was performed using an ABI 7500 Fast Real-Time PCR System (Applied Biosystems, Foster City, CA, USA). The qRT-PCR primer sets for tyrosinase and Tyrp-1 were purchased from Applied Biosystems, and TaqMan Gene Expression Assay kits (Applied Biosystems) were used for amplification. Target gene expression was normalized to that of the housekeeping gene encoding ribosomal protein lateral stalk subunit P0 (RPLP0). Relative quantization was performed using the comparative ∆∆Ct method according to the manufacturer’s instructions.
10
Gene Expression Analysis of Lipid Metabolism
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Total RNA samples were prepared using Trizol reagent (Invitrogen). RNA samples were purified using a Qiagen RNeasy kit (Qiagen, Valencia, CA, USA). Sample RNA concentration was measured spectrophotometrically at 260/280 nm. RNA sample integrity was validated using an Agilent 2100 BioAnalyzer (Agilent Technologies, Santa Clara, CA, USA). For cDNA synthesis, total RNA samples were reversely transcribed using the Superscript Reverse Transcriptase (RT) II Kit (Invitrogen). TaqMan Universal Master Mix II and Q-RT-PCR primer sets (Applied Biosystems, Foster City, CA, USA) were used to determine the transcription levels of acetyl-CoA carboxylase beta (ACACB, Hs00163715_m1), fatty acid desaturase 1 (FADS1, Hs01096545_m1), 3-hydroxy-3-methylglutaryl-CoA synthase 1 (HMGCS1, Hs00940429_m1) and lipoprotein lipase (LPL, Hs00173425_m1). Human glyceraldehyde-3-phosphate dehydrogenase (GAPDH, 4333764F) was used to normalize sample variations. Q-RT-PCR was performed with an Applied Biosystems 7500 real-time PCR system (Applied Biosystems). Relative gene expression was quantified using the Pfaffl method (Pfaffl et al., 2002 (link)).
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