Total RNA was extracted following the instructions of the Trizol reagent (Invitrogen, Carlsbad, CA, USA), reverse transcribed into cDNA, and amplified using the SYBR Green RT-PCR kit (Vazyme, Nanjing, China). The ABI7000 fluorescent quantitative PCR instrument (Applied Biosystems, Foster City, CA, USA) was used for amplification. The reaction procedure included 50 °C for 2 min, 95 °C for 10 min, 40 cycles of 95 °C for 30 sec, and 60 °C for 30 sec. The melting curve was graphed, and the final data analyzed using the formula 2−ΔΔCt. Primer sequences were 5ʹ-GGGTTTCTCGTGTGGCGCTACCTTC-3ʹ (forward) and 5ʹ-TGCCGACTTCCGCTTCCACTTGC-3ʹ (reverse) for UHRF1.
Sybr green rt pcr kit
Manufactured by Vazyme
Sourced in China
The SYBR Green RT-PCR kit is a real-time reverse transcription PCR (RT-PCR) reagent designed for the detection and quantification of RNA targets. The kit includes all the necessary components for the RT-PCR reaction, including a SYBR Green I-based detection system.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (3 mentions)
Primescript rt reagent kit, by Takara Bio (2 mentions)
Trizol reagent, by Tiangen Biotech (1 mentions)
Nanodrop 2000, by Thermo Fisher Scientific (1 mentions)
Gdna eraser, by Takara Bio (1 mentions)
All in one mirna rt qpcr detection kit, by GeneCopoeia (1 mentions)
Ribozol, by Merck Group (1 mentions)
Rna extraction kit, by Vazyme (1 mentions)
Primescript rt regent kit, by Takara Bio (1 mentions)
Lightcycler 480 2 system, by Roche (1 mentions)
Trizol, by Thermo Fisher Scientific (1 mentions)
Primescript rt master mix, by Takara Bio (1 mentions)
All in one mirna qrt pcr detection kit, by GeneCopoeia (1 mentions)
9 protocols using sybr green rt pcr kit
1
Quantifying UHRF1 gene expression
2
Diurnal Transcriptional Dynamics of PH13
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To investigate the transcriptional dynamics of PH13 in different genotypes and assess the co-expression of PH13 and GmCOP1s, we grew W82H1 and TL1H3 separately under long-day conditions for 20 days. The second fully expanded trifoliolate leaves were harvested every 4 hours over a day. Two pairs of primers were designed for qPCR to detect the expression levels of different PH13 alleles, one targeting exon 3, and the other targeting the 3’UTR. Total RNA was extracted using Trizol Reagent (TIANGEN), and then treated with DNase. A reverse transcription kit (TransGen Biotech) was used to synthesized cDNA from 3 μg of total RNA in a 20 μl reaction. RT-qPCR was performed on 384-well optical plate using the ABI Q7 equipment and the SYBR Green RT-PCR kit (Vazyme Biotech). All primers used for the indicated genes were listed in the Supplementary Data 8 . Three independent biological replicates were carried out for each sample.
3
Quantitative Analysis of miRNA and mRNA Levels
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Ribozol (Sigma-Aldrich; Merck KGaA) was used to isolate total RNAs from tissues and cells. miRNAs were harvested by precipitating RNAs with 85% ethanol. RNA concentrations were measured using NanoDrop 2000 (Thermo Fisher Scientific. Inc.) and genomic DNAs were digested using gDNA eraser (Takara Bio, Inc.). RNA samples with an OD 260/280 ratio ~2.0 (pure RNA) were reverse transcribed using the PrimeScript RT Reagent kit (Takara Bio, Inc.). The detailed workflow was as follows: 37°C For 15 min and 85°C for 5 sec then maintained at 4°C. Then the qPCR assays performed using SYBR-Green RT-PCR kit (Vazyme Biotech Co., Ltd.) to measure the expression levels of GABPB1-IT1 and PEDF mRNA. The detailed workflow was as follows: 95°C For 10 min, 40 cycles of 95°C for 15 sec and 60°C for 45 sec, final annealing at 72°C for 10 min. The expression levels of mature miR-93 were measured using the All-in-One™ miRNA RT-qPCR Detection kit (GeneCopoeia, Inc.). U6 and GAPDH were used as the endogenous controls of miR-93 and PEDF, respectively. PCR reactions were performed in triplicate and Cq values were processed using the Δ-Δq (ΔΔCq) method (18 (link)).
4
RNA Extraction and qRT-PCR Analysis
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TRIzol reagents (135306; Ambion) were used to extract RNA from BATs. We used 4× gDNA Wiper Mix to rinse genomic DNA, and used 5× HisScript II qRT SuperMix II form RNA reverse-transcription. Relative mRNA levels were determined using the SYBR Green RT-PCR Kit (Q311; Vazyme Biotech Co., Ltd., Nanjing, China) and the Realplex Real Time PCR Thermocycle Instrument (Realplex 4; Eppendorf, Westbury, NY, USA). Primer sequences we used in this study were as follows: UCP-1: 5′-A C T G C C A C A C C T C C A G T C A T T-3′, 5′-C T T T G C C T C A C T C A G G A T T G G-3′; β-actin: 5′-A G G C C C A G A G C A A G A G A G G T A-3’, 5’-G G G G T G T T G A A G G T C T C A A A C A-3′ (32 (link)). Real-time PCR data were analyzed using the 2–∆∆Ct method. Ribosomal L32 mRNA levels were used as the internal control.
5
Zebrafish Transcriptome Analysis
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Total RNA was isolated from zebrafish embryos and cells using an RNA Extraction Kit following the manufacturer’s instructions (RX112, Vazyme, Nanjing, China). cDNA templates were synthesized from RNA using the PrimeScript™ RT Regent Kit (RR036A, Takara Bio, Shiga, Japan). qRT-PCR analysis was performed with a SYBR Green RT-PCR Kit (Q712-02, Vazyme, Nanjing, China) and a Roche Light Cycler 480 II system (Roche, Switzerland). The sequences of primers used are listed in Supplementary Table 12 . GAPDH or gapdh was used as the internal control in all the experiments.
6
Soybean Leaf RNA Extraction and qRT-PCR
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Total RNA was extracted from 13-day old soybean leaves under 12 h light/12 h dark conditions using Trizol (Invitrogen, 15,596–026, California, United States). Quantitative real-time polymerase chain reaction (qRT-PCR) was performed in 384-well optical plates on the QuantStudio 7 Flex (Massachusetts, United States) using SYBR Green RT-PCR kit (Vazyme, Q221-01, Nanjing, China). The GmActin11 was used as an internal control. Three independent biological replicates and two mechanical replicates were evaluated. Related genes and primer lists are presented in Supplementary Table S3 .
7
Comprehensive RNA Extraction and Gene Expression Analysis
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The RNA-Quick Purification Kit (YISHAN Biotechnology., LTD, China) was utilized to extract total RNA, and PrimeScript RT Master Mix (TaKaRa, Japan) was employed for the synthesis of cDNA. The SYBR Green RT-PCR kit (Vazyme, China) was used to conduct fluorescence qRT-PCR assays. Relative gene expression was identified using the 2−ΔΔCt method and normalized to GAPDH. Primers for TASL, STAT3, BCL-2, BAX, TNF-α, and IL6 were designed by TSINGKE (Beijing, China).
8
Quantifying mRNA Expression in Adipose Tissues
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Total RNAs from the BAT or WAT were extracted using TRIzol reagents (135306; Ambion, Foster City, CA, United States). Then 4× gDNA Wiper Mix was used to rinse genomic DNA. Subsequently, the mRNAs were reverse-transcribed into cDNA by using 5× HisScript II qRT SuperMix II according to the manufacturer’s protocol. Relative mRNA levels were determined using the SYBR Green RT-PCR Kit (Q311; Vazyme Biotech Co., Ltd., Nanjing, China) and the Realplex Real Time PCR Thermocycle Instrument (Realplex 4; Eppendorf, Westbury, NY, United States). Real-time PCR data were analyzed with the comparative CT method. β-actin and GAPDH mRNA levels were used as an internal control. Primers used in this study for the qPCR analysis were as followed: β-actin: 5′-AGGCCCAGAGCAAGAGAGGTA-3′, 5′-GGGGTGTTGAAGGTCTCAAACA-3′; GAPDH: 5′-ACAGTCCATGCCATCACTGC-3′, 5′-GATCCACGACGGACACATTG-3′ (Ruigroka et al., 2018 (link)); Ucp-1: 5′-ACTGCCACACCTCCAGTCATT-3′, 5′-CTTTGCCTCACTCAGGATTGG-3′ (Zhou et al., 2014 (link)).
9
Quantifying Gene and miRNA Expression
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PrimeScript RT Reagent Kit (Catalog RR047A, Takara) was used to reverse transcribe total RNAs into cDNA. SYBR Green RT-PCR Kit (Catlog Q111, Vazyme Biotech) was used to prepare qPCR reactions. The expression levels of MAFG-AS1 and SphK1 were determined with GAPDH as an endogenous control. Measurement of the expression levels of mature miR-125b-5p was performed using All-in-One™ miRNA qRT-PCR Detection Kit (Catalog QP015, Genecopoeia) with all steps completed following the manufacturer’s instructions. The Ct values of 3 replicate reactions were processed using Ct (ΔΔCt) method to calculate fold changes of gene expression levels across samples. Primer sequences were: 5′-CGAAGATCTCCTCACCTCCC-3′ (forward) and 5′-TTAAAGCCGGTCGTGGAGAT-3′ (reverse) for MAFG-AS1; 5′-AGCTTCCTTGAACCATTATGCTG-3′ (forward) and 5′-AGGTCTTCATTGGTGACCTGCT-3′ (reverse) forSphK1; 5′-GGTGATTGTGGTGAGCGTGTT-3′ (forward) and 5′-AGGCCACATCAATGAGGAAGA-3′ (reverse) for GAPDH. Forward primer of miR-125b-5p was 5′-TCCCTGAGACCCTAACTTG-3′. Universal reverse primer and U6 forward primer were from the kit.
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