Mastercycler ep realplex rt pcr system

The 12 pairs of primers were designed by Nanjing DynaScience Biotechnology Co., Ltd. (www.tsingke.net, accessed on 15 March 2023). Total RNA was extracted by Trizol method; total RNA (1 μg) was reverse-transcribed with reverse transcriptase Beijing Adera Biological Co., Ltd. (www.aidlab.cn, accessed on 15 March 2023) to obtain cDNA. BeastarTM Real-time PCR Master Mix SYBR Green (DBI Bioscience, https://www.xinghanbio.com, accessed on 15 March 2023) was used as PCR Mix, and the Mastercycler EP Realplex RT-PCR system (Eppendorf, https://www.eppendorf.com, accessed on 15 March 2023) to detect expressed gene levels. To determine the differences in relative changes among samples in each experiment, the Ct value of β-actin was used for data normalization, and the 2^−ΔΔCt method [54 (link)] was used to calculate the relative changes.

As previously described, qRT-PCR was conducted for quantification of cytokines mRNA expression and validation of the selected lncRNAs and mRNAs in the hippocampal tissue [25 (link), 41 (link)]. The hippocampus of the mouse was collected on day 3 after CA/CPR procedure. With TRIzol reagent (Invitrogen, United States), the total hippocampal RNA was extracted, followed by reversely transcribed utilizing a PrimeScript RT reagent Kit with gDNA Eraser (PerfectReal Time) (Bio-Rad, USA) to synthesize cDNA in accordance with the manufacturer’s instructions. The Qubit® Assay Kit (Life Technologies, United States) and Nano 6000 Assay Kit (Agilent Technologies, United States) were used to measure the concentration of total RNA and assess RNA integrity, respectively. qRT-PCR was conducted through a Mastercycler ep realplex RT-PCR system (Eppendorf, NY). 18S mRNA was used as a reference. The primer pairs for this study were displayed in Table 2.

Total RNA was prepared using RNAiso Plus (Takara, code no. 9108) according to the manufacturer's protocol. Total RNA was transcribed into cDNA using a Prime Script TM RT Master Mix (Takara, code no. RR036A) according to the manufacturer's instructions. RT-PCR was carried out on a Master cycle Rep Realplex RT-PCR system (Eppendorf, Germany) using SYBR Premix Ex Taq (Tli RNase H Plus; Takara, Code No. RR420A) to determine the expression of HMGCR, ACAT2, ApoB, CYP7A1, FAS, and ACC in rat liver tissue. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal control. The primers for RT-PCR are presented in Table 1. PCR was carried out in duplicate by incubating the reaction mix at 95°C for 2 min followed by 40 cycles of 95°C for 15 s, 60°C for 20 s, and 72°C for 30 s. The relative levels of target mRNAs were determined using the 2^−ΔΔCt method and were normalized to the GAPDH mRNA signals in each sample.