Total RNA was extracted from the epididymal adipose tissue using an Easy‐Blue kit (Intron Biotechnology Inc) according to the protocol provided by the manufacturer. Then, total RNA was quantified with a NanoDrop‐2000 (Thermo Fisher Scientific). cDNA was synthesized (an equal amount of total RNA) with the Moloney murine leukemia virus transcriptase and Oligo (dT) 15 primers (Promega) using a Life Touch thermal cycler (Life Eco, Bioer Technology). The program was set for 1 hr of initiation at 42°C, followed by 10 min of incubation at 95°C and 10 min at 4°C. RT‐PCR was performed using the QuantiTect SYBR Green PCR kit (Qiagen), according to the manufacturer's instructions. cDNA was amplified for 40 cycles of denaturation (95°C for 30 s), annealing (57°C for 40 s), and extension (72°C for 40 s) using a RotorGene RG3000 real‐time PCR machine (Corbett Research). The purity of the PCR product was determined using melting curve analysis. The relative quantification of the expression of each gene was calculated using the comparative threshold cycle (Ct) method (Applied Biosystems). mRNA levels were normalized to β‐actin. Primer sequences are shown in Table 2 .
Life touch thermal cycler
Manufactured by Bioer
Sourced in China
The Life Touch thermal cycler is a laboratory instrument used for the amplification and analysis of DNA samples. It is designed to precisely control the temperature of samples during the polymerase chain reaction (PCR) process, which is a fundamental technique in molecular biology and genetics.
Automatically generated - may contain errors
Lab products found in correlation
Nanodrop 2000, by Thermo Fisher Scientific (4 mentions)
Rotor gene rg 3000 real time pcr machine, by Qiagen (2 mentions)
Easy blue kit, by iNtRON Biotechnology (2 mentions)
Quantitect sybr green pcr kit, by Qiagen (2 mentions)
Oligo dt 15 primer, by Promega (2 mentions)
Bigdye terminator cycle sequencing kit v3, by Thermo Fisher Scientific (1 mentions)
Amplitaq gold 360 master mix kit, by Thermo Fisher Scientific (1 mentions)
Abi 3130 genetic analyzer, by Thermo Fisher Scientific (1 mentions)
4 protocols using life touch thermal cycler
1
Epididymal Adipose Tissue RNA Extraction and qRT-PCR
2
Epididymal Adipose Tissue: RNA Extraction and Gene Expression Analysis
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Total RNA was extracted from the epididymal adipose tissue using an Easy-Blue kit (Intron Biotechnology Inc., Seoul, Korea) according to the protocol provided by the manufacturer. Then, total RNA was quantified with a NanoDrop-2000 (Thermo Fisher Scientific, Wilmington, DE, USA). cDNA was synthesized (0.03 μg of total RNA) with the Moloney murine leukemia virus transcriptase and Oligo (dT) 15 primers (Promega, Medison, WI, USA) using a Life Touch thermal cycler (Life Eco, Bioer Technology, Hangzhou, China). The program was set for 1 h of initiation at 42 °C, followed by 10 min of incubation at 95 °C and 10 min at 4 °C. RT-PCR was performed using the QuantiTect SYBR Green PCR kit (Qiagen), according to the manufacturer’s instructions. The cDNA (20 μL) was amplified for 40 cycles of denaturation (95 °C for 30 s), annealing (57 °C for 40 s), and extension (72 °C for 40 s) using a RotorGene RG3000 real-time PCR machine (Corbett Research, Sydney, Australia). The purity of the PCR products was determined using melting curve analysis. The relative quantification of the expression of each gene was calculated using the comparative threshold cycle (Ct) method (Applied Biosystems, Foster City, CA, USA). mRNA levels were normalized to β-actin. Primer sequences are shown in Table 2 .
3
Genomic DNA Isolation and Mitochondrial Sequencing
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We used the conventional phenol-chloroform method (Sambrook and Russell, 2001) to isolate the genomic DNA from liver or muscle tissues preserved in ca. 100% ethanol. Amplification was performed by PCR using an automated thermal cycler (Life Touch thermal cycler; Bioer Technology, Hangzhou, China). An AmpliTaq Gold 360 Master Mix kit (Applied Biosystems, Foster City, CA, USA) was used for PCR amplification. Aliquots of 0.1-0.2 μg of template DNA were added to a total volume of 20 μL of PCR mixture. The primers were M15997 and H16401 originally designed by Hirota et al. (2004) and similarly adopted by Sato et al. (2014) . The PCR conditions were as follows: initial denaturation at 94°C for 3 min, followed by 35 cycles of denaturation at 94°C for 30 s, annealing at 50°C for 30 s, extension at 72°C for 30 s, and a final extension at 72°C for 10 min. We also conducted PCR with a negative control, and confirmed no amplification on agarose gel electrophoresis. Sequencing of the PCR products was carried out using a Big-Dye Terminator Cycle Sequencing kit v3.1 (Thermo Fisher Scientific, Tokyo, Japan), followed by analyses on an ABI3130 genetic analyzer (Thermo Fisher Scientific).
4
Targeted cPCR Assays for SatDNA and kDNA
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Two cPCR methods were performed as described by Velázquez et al 21 : one targeting a 188-bp sequence from SatDNA and the other one targeting the 330-bp variable sequence of kDNA (Table 1). In addition, the amplification of a fragment of the ACTB gene was used to ensure the quality of DNA sample (Table 1). 21 Amplifications were performed in a LifeTouch thermal cycler (BIOER, Hangzhou, China), and PCR products were visualized under a UV transilluminator after agarose gel electrophoresis using 2% agarose gels that were stained with ethidium bromide.
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