The hypothalamus and pituitary were dissected from the brain to detect the mRNA expression of GnRH, Kiss-1, G protein-coupled receptor 54 (GPR54) in the hypothalamus and GnRH-R in the pituitary. The total RNA was extracted according to the instructions of the HiPure Total RNA Mini Kit (MAGEN), and the RNA concentration was measured by using a UV spectrophotometer (UV-2000, Unico, Shanghai, China). According to the instructions, reverse transcription was performed on a T100 Thermal Cycler PCR machine (Bio-Rad, USA) using the Reveraid First Strand cDNA Synthesis kit and the SYBR PCR master mix. Amplification and quantification were performed on a Real-Time PCR machine (Bio-Rad, USA). The RT-RCR method was as follows: initial denaturation at 95°C for 10 min, then denaturation at 95°C for 10 s, annealing at 55°C (annealing temperature of Kiss-1/GPR54 is 60°C), and extension for 30 s with a total of 50 amplification cycles. The β-actin gene was used as the internal standard. Using the 2−ΔΔCt method to calculate the relative expression levels, the primers were synthesized by Beijing Bomad Gene Technology Co., Ltd. (Beijing, China). The primers' sequence is shown in Table 1 .
T100 thermal cycler pcr machine
Manufactured by Bio-Rad
Sourced in United States
The T100 Thermal Cycler is a programmable instrument used for the amplification of DNA sequences through the Polymerase Chain Reaction (PCR) process. It features a temperature-controlled sample block that can accommodate various sample tube formats. The T100 Thermal Cycler precisely controls the temperatures and thermal cycling parameters required for efficient DNA amplification.
Automatically generated - may contain errors
Lab products found in correlation
Uv 2000, by Unico (4 mentions)
Real time pcr machine, by Bio-Rad (4 mentions)
Reveraid first strand cdna synthesis kit, by Bio-Rad (2 mentions)
Hipure total rna mini kit, by Magen Biotechnology Co (1 mentions)
Revertaid first strand cdna synthesis kit, by Thermo Fisher Scientific (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Sybr select master mix, by Thermo Fisher Scientific (1 mentions)
4 protocols using t100 thermal cycler pcr machine
1
Gene Expression Analysis of Hypothalamic-Pituitary Axis
2
Quantitative Analysis of NLRP3 Inflammasome Genes
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RNA was isolated from the cortical tissue in the penumbra using Trizol Reagent (Invitrogen, Grand Island, NY, USA). The RNA concentration was measured using an ultraviolet spectrophotometer (UV-2000, Unico, Shanghai, China). Then, reverse transcription was performed from 2 μg RNA on a T100 Thermal Cycler PCR machine (Bio-Rad, USA) using Revert Aid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific, Waltham, WA, USA,). Quantitative PCR was performed according to the manufacturers’ instructions using SYBR Select Master MIX (K1622, Applied Biosystems, CA, USA) and a Real-Time PCR machine (Bio-Rad, USA). Expression values of the targeted genes were normalized to the corresponding expression of β-actin. The 2−ΔΔCt method was used to calculate the relative gene expression. Sequence-specific primers are listed in Table 1 .
Real-time fluorescent quantitative PCR primers
| Gene names | Forward | Reverse |
|---|---|---|
| NLRP3 | CAGCGATCAACAGGCGAGAC | AGAGATATCCCAGCAAACCTATCCA |
| ASC | GCTGAGCAGCTGCAAACGA | ACTTCTGTGACCCTGGCAATGA |
| Caspase-1 | ACTCGTACACGTCTTGCCCTCA | CTGGGCAGGCAGCAAATTC |
| IL-1β | CCCTGAACTCAACTGTGAAATAGCA | CCCAAGTCAAGGGCTTGGAA |
| GAPDH | GAACATCATCCCTGCATCCA | CCAGTGAGCTTCCCGTTCA |
3
Quantifying Hippocampal Gene Expression
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The hippocampus of five rats from each group was dissected from the brains. Based on the Hipure Total RNA mini kit instructions, the total RNA was extracted. The ultraviolet spectrophotometer (UV-2000, Unico, Shanghai, P. R. China) was used to detect the RNA concentration. We used the RevertAid first-strand cDNA synthesis kit to synthesize cDNA. According to the manufacturers' instructions, we used the cDNA and the SYBR PCR master mix to conduct the reverse transcription on the T100 Thermal Cycler PCR machine (Bio-Rad, USA). Amplification and quantitative detection were performed in a Real-Time PCR machine (Bio-Rad, USA). The RT-PCR protocol was as follows: initial denaturation for 10 min at 95 • C, then 45 cycles for 10 s at 95 • C, and finally 59 • C for 60 s. The following primers sequences for target genes were used: MR F: 5' CAAGGAGCCATCGGTGAACT 3' MR R: 5' AGAGGAGTTGGCTGTTCGTG 3' (341bp); GR F: 5' GGCTTCTGTCTAGAATATGCCTG 3' GR R: 5' GATTTGTCCCCATTATATAGCCTT 3' (156bp);
β-actin F: 5' GCACCATGAAGATCAAGATCATT 3' β-actin R: 5' TAACAGTCCGCCTAGAAGCATT 3' (172bp). The 2 -∆∆Ct method (Livak and Schmittgen, 2001) was used for relative quantitative analysis of the results.
β-actin F: 5' GCACCATGAAGATCAAGATCATT 3' β-actin R: 5' TAACAGTCCGCCTAGAAGCATT 3' (172bp). The 2 -∆∆Ct method (Livak and Schmittgen, 2001) was used for relative quantitative analysis of the results.
4
Quantifying Hippocampal Gene Expression
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The hippocampus was dissected from the brain tissue, and the total RNA was extracted according to the Hipure Total RNA mini kit instructions. The RNA concentration was measured using an ultraviolet spectrophotometer (UV-2000, Unico, Shanghai, China). Reverse transcription was performed on a T100 Thermal Cycler PCR machine (Bio-Rad, USA) using the Reveraid First Strand cDNA Synthesis kit and the SYBR PCR master mix according to the manufacturers’ instructions. Amplification and quantitative detection were performed in a Real-Time PCR machine (Bio-Rad, USA). The RT–PCR protocol was as follows: initial denaturation at 95 °C for 15 min, then 55 cycles at 95 °C for 10 s, and finally 58 °C for 30 s. Primer sequences for the genes of interest were as follows:
IDO1 F: 5′ GGATCCTTGAAGACCACCACAT 3′
IDO1 R: 5′ AAGGACCCAGGGGCTGTAT 3′
NF-κB P65 F: 5′ ATCATCGAACAGCCGAAGCA 3′
NF-κB P65 R: 5′ TGATGGTGGGGTGTGTCTTG 3′
β-actin F: 5′ CCTAGGCACCAGGGTGTG 3′
β-actin R: 5′ CGGTGAGCAGCACAGGGT 3′
The 2−ΔΔCt method was used for relative quantitative analysis of the results.
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