According to the manufacturer’s instructions, total RNA was extracted with an RNA Fast2000 kit (Fastagen, Shanghai, China). RNA was reverse transcribed using PrimeScript RT Reagent Kit with gDNA Eraser (Takara). According to the manufacturer’s instructions, the quantification of gene transcripts was performed by quantitative Real-Time PCR using TB Green (Takara) and the ABI7500Fast Real-Time PCR systems (ABI) instructions. Data were normalized by the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression level in each sample, and the 2-△△Ct method was used to calculate relative expression changes. With the help of dissociation curve analysis and the sequencing of PCR products, pairs of specific primers of each cDNA were designed and selected without any primer-dimers or unspecific amplification detected. The specific primers for individual genes were as follows: Ocilrp2, 5’-TTCTGGATACCCACGTAACTGG-3’ (sense) and 5’-TCCCCCTTGAATCTCTTTAGGAA-3’ (antisense); IL-6, 5’-TAGTCCTTCCTACCCCAATTTCC-3’ (sense) and 5’-TTGGTCCTTAGCCACTCCTTC-3’ (antisense); Nkrp1f, 5’-TTAGGTGTCCAGGGTATAAGCA-3’ (sense) and 5’-AGCACAGCCAGATTTCAGAGC-3’ (antisense); Nkrp1g, 5’-AACCCTGTGTCCTGACTCCT-3’ (sense) and 5’-CTTTGTGCCACTAACGGTGC-3’ (antisense); Gapdh, 5’-GGTGAAGGTCGGTGTGAACG-3’ (sense) and 5’-CTCGCTCCTGGAAGATGGTG-3’ (antisense).
Abi7500fast real time pcr systems
Manufactured by Thermo Fisher Scientific
Sourced in United States
The ABI7500Fast Real-Time PCR system is a thermal cycler designed for real-time PCR analysis. It is capable of performing quantitative, end-point, and allelic discrimination assays. The system features a 96-well sample block, a color touchscreen interface, and supports a variety of fluorescent chemistries and detection formats.
Automatically generated - may contain errors
Lab products found in correlation
Rnafast2000 kit, by Fastagen (1 mentions)
Tb green, by Takara Bio (1 mentions)
Primescript rt reagent kit with gdna eraser, by Takara Bio (1 mentions)
Reverse transcription reagent, by Promega (1 mentions)
Total rna miniprep kit, by Corning (1 mentions)
Qscript, by Quanta Biosciences (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
3 protocols using abi7500fast real time pcr systems
1
Quantitative Real-Time PCR Protocol
2
Quantitative Real-Time RT-PCR Analysis
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Total RNA was extracted from cells using the total RNA miniprep kit (Axygen, China) and subjected to reverse transcription with reverse transcription reagent (Promega, US). Real-time RT-PCR was performed on a ABI 7500-fast Real-time PCR systems (ABI, US). Each 20 μl qPCR reaction contained 2 μl reverse transcription sample, 10 μl TliRNaseH Plus (2×), 0.4 μl forward and reverse primers (10 μM), 0.4 μl ROX Reference Dye Ⅱ (50×), and 6.8 μl sterile purified water. Amplification conditions were: 95 ℃ for 30 s, followed by 40 cycles of 95 ℃ for 5 s and 60 ℃ for 34 s. Each reaction was performed in triplicate using GAPDH as the internal control, and the qRT-PCR data was analyzed using the 2−△△CT method.
3
Protein and Gene Expression Analysis
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Before and after treatment with indicated drugs, or transfections with the siRNAs against indicated genes, whole lysate proteins extracted from the cells were prepared and analyzed by standard immunoblot assay as previously reported 17 (link),18 (link).
For real-time quantitative PCR analysis, total RNA was extracted from cell samples using Trizol reagents (Invitrogen) following the manufacturers' instructions. RNA was reverse transcribed into cDNA using qScript (Quanta Biosciences) following the manufacturer's instructions and the reactions were run on an ABI 7500 Fast real-time PCR systems, as previously reported 17 (link). For more details seeSupplementary materials and methods.
For real-time quantitative PCR analysis, total RNA was extracted from cell samples using Trizol reagents (Invitrogen) following the manufacturers' instructions. RNA was reverse transcribed into cDNA using qScript (Quanta Biosciences) following the manufacturer's instructions and the reactions were run on an ABI 7500 Fast real-time PCR systems, as previously reported 17 (link). For more details see
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