Chemicals and reagents used in this study were purchased from the following sources: corn oil and tamoxifen from Aladdin (Shanghai, China); Taq Plus Master Mix Ⅱ, GelRed Nucleic Acid Stain, agarose gel, DNA markers, and DNA Isolation Kit from Vazyme (Nanjing, China); total RNA extraction reagent, RT-PCR kit, and SYBR Premix Ex Taq™ kit from Takara (Dalian, China); eIF3a and GAPDH antibody from Abcam (Cambridge, UK); methanol, ethanol, acetone, and acetonitrile from Sinopharm (Shanghai, China); and BCA Protein Assay Kit from Invitrogen (Grand Island, United States).
Dna isolation kit
Manufactured by Vazyme
Sourced in China
The DNA isolation kit is a laboratory equipment designed to extract and purify DNA from various biological samples. It provides a reliable and efficient method to obtain high-quality DNA for further analysis and applications.
Automatically generated - may contain errors
Lab products found in correlation
Sybr premix ex taq kit, by Takara Bio (1 mentions)
Rt pcr kit, by Takara Bio (1 mentions)
Taq plus master mix 2, by Vazyme (1 mentions)
Methanol, by Sinopharm (1 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (1 mentions)
Total rna extraction reagent, by Takara Bio (1 mentions)
Eastep super total rna extraction kit, by Promega (1 mentions)
Mirna 1st strand cdna synthesis kit, by Vazyme (1 mentions)
Chamq universal sybr qpcr kit, by Vazyme (1 mentions)
Hiscript 2 q select rt supermix kit, by Vazyme (1 mentions)
Pyromark q96 id, by Qiagen (1 mentions)
Pyro q cpg, by Qiagen (1 mentions)
5 protocols using dna isolation kit
1
Reagents and Chemicals Utilized
2
Quantifying Mitochondrial DNA Copy Number
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The relative copy number of mtDNA was quantified using nuclear DNA content as a standard. Human mtDNA Copy Number Assay Kit (Takara) was used for RT-PCR. DNA was separated from CMECs using a DNA isolation kit (Vazyme, Nanjing, China) and 20 μL of reaction mixture per PCR tube containing 1 μL forward primer, 1 μL reverse primer, 8 μL sample, and 10 μL reaction mix was prepared on ice. Following the reaction was completed, the mtDNA copy number was calculated according to the 2 ∆Ct method.
3
Quantification of miR-9, Transcripts, and Viral Genomes
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To quantify miR-9 in cells or tissues, total RNA was purified using an Eastep Super total RNA extraction kit (catalogue no. LS1040m, Promega) following the manufacturer’s protocol for retaining small RNA, reverse transcribed using a miRNA 1st Strand cDNA Synthesis Kit (catalogue no. MR101-01, Vazyme Biotech), and PCR amplified using a ChamQ Universal SYBR qPCR kit (catalogue no. Q711-02/03; Vazyme Biotech). The stem-loop primers and qPCR primers for miR-9 quantification were purchased from RiboBio. To quantify transcripts, RNA was isolated using an Eastep Super total RNA extraction kit (catalogue no.LS1040m, Promega), reverse transcribed using a HiScript II Q Select RT supermix kit (catalogue no. R233-01; Vazyme Biotech), and PCR amplified using a ChamQ Universal SYBR qPCR kit (catalogue no. Q711-02/03; Vazyme Biotech). The analyzed transcript levels were normalized to GAPDH transcript levels. To quantify viral genomes, DNA was isolated using a DNA isolation kit (catalogue no.DC102-01; Vazyme Biotech) and qPCR was conducted using the ChamQ Universal SYBR qPCR kit (catalogue no. Q711-02/03; Vazyme Biotech). Viral genome levels were normalized to mouse Adipsin gene levels. Serially diluted total DNA, total RNA, or synthetic miR-9 was used to generate standard curves. PCR primer sequences are listed in Supplementary Table 4 .
4
Quantitative Analysis of MAGE-D4 Promoter Methylation
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Genomic DNA was isolated from cells using a DNA Isolation Kit (Vazyme, China). The methylation status of the MAGE-D4 promoter in cells was quantified by the PyroMark Q96 ID pyrosequencing platform (Qiagen), which was performed by Shanghai Geneland Biotech Co., Ltd. The methylation status of each site was analyzed automatically by the accompanying software Pyro Q-CpG (Qiagen). Two regions in the core promoter were detected, which contained 18 CpG sites. Primers used in pyrosequencing were as follows: region 1: 5’-TTGGAGGAAAGGGTTTTTGTTG-3’ (forward) and 5’-CCCCATCCTATCTAAAACTAAATCCTTAC-3’ (reverse); region 2: 5’-GGTTGAGGGGTTTTTGGTGT-3’ (forward) and 5’-AAAAACTCCTATCTAAACCTTAAATC-3’ (reverse). Sequences of the MAGE-D4 promoter and pyrosequencing regions containing CpG sites are listed in Supplemental Materials
5
Quantifying Mitochondrial Content and Morphology
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For mitochondria content assay, genomic DNA was extracted from adipose tissues by DNA isolation kit (Vazyme Biotech Co., DC112). A certain amount of genomic DNA (about 100 ng) was used as templates for quantitative real‐time PCR to detect the mitochondria‐encoded gene Cox2 and nuclear‐encoded gene Cebpα levels. The mitochondrial content is shown as the ratio of the mitochondrial DNA level relative to nuclear DNA level. For measuring mitochondrial diameter, Image J software was used to quantify the transverse diameter of each mitochondrion in the EM images. 60 mitochondria were measured in each group and the average diameter was shown. Cristae abundance was estimated by the ratio of inner to outer mitochondrial membrane perimeter.[57 ] Thirty mitochondria were analyzed using Image J.
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