For the qRT-PCR analysis, 1 µg of total RNA was reverse transcribed using the RT reagent Kits with gDNA Eraser (Takara, Dalian, China), according to the manufacturer’s protocol. The qRT-PCR was performed on a StepOnePlus Real-Time PCR System (Life Technologies, Gaithersburg, MD, USA), according to standard methods using Fast Start Universal SYBR Green Master (ROX) (Roche, Mannheim, Germany).
Rt reagent kits with gdna eraser
Manufactured by Takara Bio
Sourced in China
The RT reagent Kits with gDNA Eraser are a set of reagents designed for reverse transcription and genomic DNA removal. The kits provide the necessary components for the conversion of RNA into cDNA, as well as the elimination of genomic DNA contamination from RNA samples.
Automatically generated - may contain errors
Lab products found in correlation
Steponeplus real time pcr system, by Thermo Fisher Scientific (3 mentions)
Faststart universal sybr green master rox, by Roche (3 mentions)
Lightcycler 480 sybr green 1 master, by Roche (1 mentions)
Cfx connect real time pcr detection system, by Bio-Rad (1 mentions)
Abi 7300, by Thermo Fisher Scientific (1 mentions)
5 protocols using rt reagent kits with gdna eraser
1
qRT-PCR Analysis of Total RNA
2
qRT-PCR Transcription and Detection
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For the qRT-PCR study, 1 μg of total RNA was reverse transcribed according to the manufacturer's instructions using the RT reagent Kits with gDNA Eraser (Takara, Dalian, China). The qRT-PCR was carried out using a StepOnePlus Real-Time PCR System (Life Technologies, Gaithersburg, MD, USA) using Fast Start Universal SYBR Green Master (ROX) according to routine procedures (Roche, Mannheim, Germany).
3
RT-qPCR Validation of RNA-seq Findings
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To verify the results of high-throughput RNA-seq, RT-qPCR was conducted. For the RT-qPCR analysis, 1 μg of total RNA was reverse transcribed using the RT reagent Kits with gDNA Eraser (Takara, Dalian, China) according to the manufacturer’s protocol. Six DE lncRNAs and four DEGs were chosen for RT-qPCR validation. Primer Premier 5.0 software was adopted to design the gene-specific primers (Additional file 7 : Table S7).
The reaction of RT-qPCR was performed with CFX Connect Real-Time PCR Detection System (BIO-RAD, USA) according to standard methods using LightCycler@ 480 SYBR Green I Master (Roche, USA). 18 s rRNA was used as an internal control [44 (link)]. All experiments were performed in triplicate. The amount of target molecules relative to the control was calculated by using the 2-ΔΔCt method [61 (link)]. The results of RT-qPCR data were presented as mean ± standard error of the mean (SEM) and were calculated by SPSS 22.0 (SPSS Inc., Chicago, IL, USA). Differences between the control and experimental treatments were analyzed by one-way analysis of variance (ANOVA) through Dunnett’s multiple comparison. The level of statistical significance was set at P < 0.05, and highly significance at P < 0.01.
The reaction of RT-qPCR was performed with CFX Connect Real-Time PCR Detection System (BIO-RAD, USA) according to standard methods using LightCycler@ 480 SYBR Green I Master (Roche, USA). 18 s rRNA was used as an internal control [44 (link)]. All experiments were performed in triplicate. The amount of target molecules relative to the control was calculated by using the 2-ΔΔCt method [61 (link)]. The results of RT-qPCR data were presented as mean ± standard error of the mean (SEM) and were calculated by SPSS 22.0 (SPSS Inc., Chicago, IL, USA). Differences between the control and experimental treatments were analyzed by one-way analysis of variance (ANOVA) through Dunnett’s multiple comparison. The level of statistical significance was set at P < 0.05, and highly significance at P < 0.01.
4
Differential Expression Analysis of lncRNAs and Genes
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Transcript abundance was measured by FPKM using Cuffdiff (version 2.1.1) [24 (link)]. The FPKMs of the protein-coding genes in each sample were computed by summing the FPKMs of the transcripts in each gene group. Differential expression analysis of the groups was performed using the DEGseq packages (1.10.1) [25 (link)]. LncRNAs or protein-coding genes with a false discovery rate (FDR) <5% and an absolute value of log2 (fold change) >1 were assigned as differentially expressed.
For the qRT-PCR analysis, 1 μg of total RNA was reverse transcribed using the RT reagent Kits with gDNA Eraser (Takara, Dalian, China) according to the manufacturer’s protocol. QRT-PCR was performed on a StepOnePlus Real-Time PCR System (Life Technologies, USA) according to standard methods using Fast Start Universal SYBR Green Master (ROX) (Roche, Mannheim, Germany). Five differentially expressed lncRNAs and six DEGs were chosen for qRT-PCR validation, including a muscle structural gene myosin heavy chain 7 (MYH7) and myogenin (MYOG). Comparative quantification of each gene was normalized to hypoxanthine phosphoribosyltransferase 1 using the 2−ΔΔCt method. All experiments were performed in triplicate.
For the qRT-PCR analysis, 1 μg of total RNA was reverse transcribed using the RT reagent Kits with gDNA Eraser (Takara, Dalian, China) according to the manufacturer’s protocol. QRT-PCR was performed on a StepOnePlus Real-Time PCR System (Life Technologies, USA) according to standard methods using Fast Start Universal SYBR Green Master (ROX) (Roche, Mannheim, Germany). Five differentially expressed lncRNAs and six DEGs were chosen for qRT-PCR validation, including a muscle structural gene myosin heavy chain 7 (MYH7) and myogenin (MYOG). Comparative quantification of each gene was normalized to hypoxanthine phosphoribosyltransferase 1 using the 2−ΔΔCt method. All experiments were performed in triplicate.
5
qRT-PCR Analysis of mRNA and lncRNA
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For the qRT-PCR analysis, 1 μg total RNA was reverse transcribed using RT reagent kits with gDNA Eraser (Takara, China) according to the manufacturer’s protocol. Real-time PCR was performed on an ABI 7300 (Applied Biosystems, Foster City, CA, U.S.A.) with Fast Start Universal SYBR Green Master (ROX) (Roche, Mannheim, Germany). The following program was used: 95°C for 5 min, followed by 40 cycles at 95°C for 10 s, 60°C for 30 s, and 72°C for 30 s. Primers for mRNAs and lncRNAs are shown in Supplementary Table S1. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal reference to normalize target gene expression. All experiments were performed in triplicate.
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