Evo m mlv reverse transcriptase kit

Peripheral blood monocytes (PBMCs) were isolated from the blood samples of AD and HC using Ficoll solution (Solarbio, Beijing, China). Total RNA was extracted from the PBMCs using TRIzol reagent (Takara, Kyoto, Japan) according to the manufacturer’s protocol. Then reverse transcription reactions were performed using 500 ng RNA and an Evo M-MLV reverse transcriptase kit (Accurate Biotechnology, Hunan, China) according to instructions. SYBR Green Pro Taq HS Kit (Accurate Biotechnology, Hunan, China) and 0.4 μmol of each primer pair were used to amplify the cDNA, then evaluated in an ABI 7500 real-time PCR system (Applied Bioscience, Foster City, CA, United States). The results were analyzed using the 2^–ΔΔCt method and expressed as the ratio of the internal control, ACTIN. The primer sequences used for the real-time PCR analysis are available in Table 2.

Patients (≥18 years old; IA, n = 12) who were diagnosed with IA at Xiangya Hospital of Central South University were recruited for this research between December 2021 and June 2022. Patients with other diseases or complications were excluded. In total, 12 healthy controls who were gender- and age-matched were also selected.
Peripheral blood monocytes (PBMCs) were isolated from the blood samples of IA and healthy persons using Ficoll solution (Solarbio, Beijing, China). Total RNA was extracted from the PBMCs using TRIzol reagent (Takara, Kyoto, Japan) according to the manufacturer’s protocol. Then, reverse transcription reactions were performed using 500 ng RNA and an Evo M-MLV reverse transcriptase kit (Accurate Biotechnology, Hunan, China) according to the manufacturer’s instructions. A SYBR Green Pro Taq HS Kit (Accurate Biotechnology, Hunan, China) and 0.4 μmol of each primer pair were used to amplify the cDNA, which was then evaluated in an ABI 7500 real-time PCR system (Applied Bioscience, Foster City, CA, United States). The results were analyzed using the 2^–ΔΔCt method and expressed as the ratio of the internal control, GAPDH. The primer sequences used for RT–qPCR are available in Supplementary Table 1.

Total RNA was extracted using RNAiso™ Plus (Takara, Japan). First-strand cDNA was synthesized from 1 μg total RNA by the Evo M-MLV reverse transcriptase kit (Accurate Biology, China). The qRT-PCR assays were performed using the SYBR® Green Pro Taq HS qPCR kit (Accurate Biology, China) in a Roche LightCycler®96 system. The PCR procedures were as follows: 95 °C for 30 s, then followed by 40 cycles of 95 °C for 5 s, 60 °C for 30 s, and the final step was dissociation stage. OsActin was used as an internal control and the data analysis was referred to 2^−△△Ct algorithm. Three biological replicates were used and the related primers were listed in Additional file 10: Table S1.