Stepone rt pcr thermocycler

The mRNA levels of MCP-1 and MIP-2 were evaluated by qRT-PCR. Total RNA was extracted from kidney tissues using TRIpure Total RNA Extraction Reagent (ELK Biotechnology, Wuhan City, China) following the manufacturer’s protocol. First-strand cDNA was synthesized using the EntiLink™ 1st Strand cDNA Synthesis Kit (ELK Biotechnology, Wuhan City, China). Target gene expression levels were measured with EnTurbo™ SYBR Green PCR SuperMix (ELK Biotechnology, Wuhan City, China) using a StepOne™ RT-PCR thermocycler (Life Technologies, Massachusetts, USA). The mRNA levels of MCP-1 and MIP-2 were normalized to the β-actin mRNA level in the same sample and calculated using the Delta-Delta-CT method. The primer sequences were as follows: MCP-1, forward: 5′-GGCCTGTTGTTCACAGTTGCT-3′; reverse, 5′-GCCGACTCATTGGGATCATC-3′; MIP-2, forward: 5′-GTCAATGCCTGACGACCCTAC-3′, reverse: 5′-CCTTCCCAGGTCAGTTAGCCT-3′; and β-actin, forward: 5′- CGTTGACATCCGTAAAGACCTC-3′, reverse: 5′-TAGGAGCCAGGGCAGTAATCT-3′.

Total mRNA was extracted from liver tissues using a TRIpure reagent (ELK Biotechnology) according to the manufacturer's protocols. The synthesis of first-strand cDNA was performed using M-MLV Reverse Transcriptase (ELK Biotechnology). The expression levels of the target genes were measured with EnTurbo™ SYBR Green PCR SuperMix (ELK Biotechnology) using a StepOne™ RT-PCR thermocycler (Life Technologies). The mRNA level of each gene was normalized to the β-actin mRNA level of the same sample using the delta-delta CT method. All RT-PCR primer sequences are listed as follows: IL-1β 5′-ATGAAAGACGGCACACCCAC-3′ and 5′-GGTGCTGATGTACCAGTTGGG-3′; IL-6 5′-GCCAGAGTCATTCAGAGCAAT-3′ and 5′-CTTGGTCCTTAGCCACTCCT-3′; TNF-α 5′-CACCACGCTCTTCTGTCTACTG-3′ and 5′-GCTACGGGCTTGTCACTCG-3′; and β-actin 5′-CGTTGACATCCGTAAAGACCTC-3′ and 5′-TAGGAGCCAGGGCAGTAATCT-3′.