Total RNA was extracted using Trizol (TransGene Biotech, Beijing, China) and then transcribed into cDNA using TranScript All-in-One First-Strand cDNA Synthesis SuperMix (TransGene Biotech), as instructed by the manufacturer. Real-time PCR was performed with an Eppendorf Realplex PCR system using TransStart Tip Green qPCR SuperMix (TransGene Biotech). The expression was normalized to the expression of the housekeeping gene GAPDH. The primer sequences used in the experiment are shown in Table 1 .
Realplex pcr system
Manufactured by Eppendorf
Sourced in United States
The Realplex PCR system is a real-time PCR instrument designed for accurate, reliable, and efficient nucleic acid amplification and quantification. It provides precise temperature control and optical detection capabilities to enable sensitive and reproducible qPCR analysis.
Automatically generated - may contain errors
Lab products found in correlation
Nanodrop 2000, by Thermo Fisher Scientific (6 mentions)
Trizol, by Thermo Fisher Scientific (5 mentions)
Tri reagent, by Merck Group (4 mentions)
Dynamo flash sybr green master mix, by Thermo Fisher Scientific (4 mentions)
Superscript 3 reverse transcriptase, by Thermo Fisher Scientific (3 mentions)
Cdna synthesis kit, by Thermo Fisher Scientific (3 mentions)
Sybr green master mix, by Thermo Fisher Scientific (3 mentions)
Revertaid first strand cdna synthesis kit, by Thermo Fisher Scientific (3 mentions)
Transstart tip green qpcr supermix, by Transgene (2 mentions)
Transcript all in one first strand cdna synthesis supermix, by Transgene (2 mentions)
Mir xtm mirna first strand synthesis, by Takara Bio (2 mentions)
Tb green qrt pcr kit, by Takara Bio (2 mentions)
Cdna reverse transcription kit, by Thermo Fisher Scientific (2 mentions)
Sybr green master mix, by Promega (2 mentions)
Trizol, by Transgene (1 mentions)
Trizol reagent, by Transgene (1 mentions)
Sybr green dye, by Thermo Fisher Scientific (1 mentions)
Mesa green master mix, by Eurogentec (1 mentions)
Sigmaplot 10, by Grafiti LLC (1 mentions)
Sybr green premix ex taq, by Takara Bio (1 mentions)
17 protocols using realplex pcr system
1
RNA Extraction and qPCR Analysis
2
Total RNA Isolation and Real-Time PCR
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Total RNA isolation was performed using TRIzol reagent (TransGene Biotech, China). A total of 1 μg of total RNA was reverse transcribed using TranScript All-in-One First-Strand cDNA Synthesis SuperMix (TransGene Biotech) according to the instructions of the manufacturer. Real-time PCR was performed with an Eppendorf Realplex PCR system using TransStart Tip Green qPCR SuperMix (TransGene Biotech). Primers used in the qPCR reactions, which were all from PrimerBank (Wang et al., 2012) and synthesized by Huada Gene Technology Co., Ltd. (Shenzhen, China), were presented in Table 1.
3
Quantitation of mtDNA Copy Number
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Total DNA extraction was performed with a DNA kit (Tiangen Biotech). The quantitation of the mtDNA per nuclear genome used in this study was performed as previously described33 (link)34 (link). In brief, with real-time PCR analysis, the NADH dehydrogenase subunit 1 (ND1), which represents the mtDNA, was normalized to the Pecam gene on chromosome 6, which represents nuDNA. The synthetic oligonucleotide primer sequences for ND1 are 5′-GTC CTC CTA ATA AGC GGC TCC T-3′ (coding sense) and 5′-GAA TGG TCC TGC GGC GTA TTC-3′ (coding antisense), and the sequences of Pecam are 5′-CTA TGG CGG ACA CCT TCC TG-3′ (coding sense) and 5′-TTC TAG GCC TTG GGT GGT CT-3′ (coding antisense). Real-time PCR was performed using SYBR Green dye (Thermo Scientific) with the Eppendorf Realplex PCR system. The relative copy number differences were quantified by analysing the difference in threshold amplification between mtDNA and nuDNA (ΔΔCt method).
4
Quantitative Real-Time PCR Analysis of Gene Expression
5
DAF-16 Promoter Enrichment Analysis
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The enrichment of DAF-16 on the promoter regions of known or newly identified genes was determined by quantitative real time PCR (qRT-PCR) using the Mesagreen MasterMix (Eurogentec, Belgium) and Realplex PCR system (Eppendorf, USA) according to manufacturer's specifications. The list of primers are provided in Table S6 . The relative enrichment in daf-2(−) was determined after normalization with input and then compared with input-normalized daf-16(−);daf-2(−). Fold change was calculated using ΔΔCT method [36 (link)] and statistical analysis was performed using SigmaPlot 10.0 (Systat software, USA).
6
Peripheral Blood miRNA and mRNA Quantification
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1μg of RNA isolated from peripheral blood was reverse transcribed and used for miRNA quantification using Mir-X TM miRNA First-Strand Synthesis and TB Green qRT-PCR kit (Takara Bio Inc.). U6 was used as an internal control. For mRNA quantification, about 1μg of RNA isolated from peripheral blood was converted to cDNA with the cDNA Reverse Transcription Kit (Applied Biosystems, USA).
Quantitative RT-PCR (qRT-PCR) was carried out on RealPlex PCR system (Eppendorf, USA) with was performed using GraphPad 7.0. The primers used are enlisted in Supplementary Table 1.
Quantitative RT-PCR (qRT-PCR) was carried out on RealPlex PCR system (Eppendorf, USA) with was performed using GraphPad 7.0. The primers used are enlisted in Supplementary Table 1.
7
RNA Extraction and Quantitative RT-PCR
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Total RNA was extracted using TRIzol reagent (Invitrogen), and RNA concentration was measured using a spectrophotometer (Bio-Rad, USA). The Revert Aid First Strand cDNA Synthesis Kit (Thermo Scientific, USA) was used to synthesize complementary DNA from corresponding RNA by reverse transcription, and SYBR Green Premix Ex Taq (Takara, Japan) was used to perform quantitative RT-PCR on a realplex PCR system (Eppendorf), with glyceraldehyde 3-phosphate dehydrogenase as the control gene. Differences in messenger RNA (mRNA) expression were assessed based on the comparative cycle threshold method. The primers used were listed in the Supplementary Table 1 .
8
Real-Time PCR Analysis of Gene Expression
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Total RNAs were isolated from cells or tissues using the TRI reagent (MilliporeSigma) and converted into complementary DNA using a complementary DNA synthesis kit (Applied Biosystems, Foster City, CA). Real-time PCR analysis was performed using SYBR Green Master Mix (Promega, Madison, WI) in an Realplex PCR system (Eppendorf North America, Hauppauge, NY). Transcript levels were analyzed with the 2−delta delta cycle threshold method, and quantification was normalized to the internal control gene PPIA. Primer sequences of the genes used in this work are described in Table 2 .
9
Comprehensive RNA Transcriptome Analysis
10
Transcriptome Analysis of C. elegans
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Eggs were synchronized by overnight starvation. These L1s were then allowed to grow till YA stage. YA worms were collected in M9 buffer after washing thrice to get rid of bacteria and then frozen in Trizol (Invitrogen, USA). The frozen worms were passed through two freeze-thaw cycles and lysed by vigorous vortexing. RNA was extracted by phenol:chloroform:isoamyl alcohol followed by ethanol precipitation. RNA was dissolved in DEPC-treated MQ water and denatured at 65⁰C for 10 min. The concentration of the RNA was then determined using NanoDrop 2000 (Thermo Scientific, USA). The integrity of the RNA was checked by electrophoresis on a denaturing formaldehyde gel.
First strand cDNA was synthesized using 2.5 μg RNA, employing Superscript III Reverse Transcriptase (Invitrogen, USA). Quantitative real time PCR (qRT-PCR) reaction was set up using DyNAmo Flash SYBR Green master mix (Thermo Scientific, USA) in a Realplex PCR system (Eppendorf, USA), according to manufacturer's specifications. The gene expression was represented as relative fold change determined after normalizing the ΔCt values to actin, the housekeeping gene.
Unpaired two-tailed t-test was applied as statistical analysis by using GraphPad Prism (GraphPad Software, La Jolla California).
First strand cDNA was synthesized using 2.5 μg RNA, employing Superscript III Reverse Transcriptase (Invitrogen, USA). Quantitative real time PCR (qRT-PCR) reaction was set up using DyNAmo Flash SYBR Green master mix (Thermo Scientific, USA) in a Realplex PCR system (Eppendorf, USA), according to manufacturer's specifications. The gene expression was represented as relative fold change determined after normalizing the ΔCt values to actin, the housekeeping gene.
Unpaired two-tailed t-test was applied as statistical analysis by using GraphPad Prism (GraphPad Software, La Jolla California).
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