Abi prism 7900ht real time pcr system

Different depots of adipose tissues, including subcutaneous WAT, epididymal WAT and brown AT, as well as heart, kidney and liver tissues were harvested and quickly frozen in liquid nitrogen for future use. For the total RNA extraction, a combination of Trizol (Invitrogen, Carlsbad, CA) reagents and the RNeasy RNA extraction kit (#74106, Qiagen, Valenica, CA) was utilized. Briefly, after homogenizing the tissues by using a TissueLyser (Qiagen), the RNAs were isolated following the protocol from the RNeasy kit. Then the quality and concentration of the RNA were determined through the Nanodrop Spectrophotometer (N1-1000, Thermo Scientific, Wilmington, DE). A total of 1 μg RNA underwent subsequently reverse transcriptional reactions with an iScript cDNA synthesis kit (#170–8891, Bio-Rad Laboratories, Inc., Hercules, CA). cDNAs were stored and utilized for further qPCR analysis of relative gene expressions. Briefly, we utilized the SYBR Green PCR Master Mix (#4309155, Life Technologies, Carlsbad, CA) to perform the qPCR reactions, and the experiments were conducted on an ABI Prism 7900HT Real-Time PCR System (Applied Biosystems, Foster City, CA).​ All the primer sequences in this study were validated in previous studies and are listed in Supplementary file 1, and Gapdh was used as the internal control.

All litters were randomly divided into saline- or poly I:C-treated groups. From PD 2 to 6, mice were injected s.c. daily with either pyrogen-free saline or poly I:C (Sigma–Aldrich) at a dose of 5 mg/kg^{11 (link)}. Neonatal mice were decapitated 2 or 24 h after the final treatment with saline or poly I:C and their brains were removed. Total RNA was isolated using the RNeasy Plus Mini Kit (Qiagen, Hilden, Germany) from each mouse (2 h after the final treatment with saline n = 6 or poly I:C n = 6, 24 h after the final treatment with saline n = 6 or poly I:C n = 6). Total RNA isolated from the prefrontal cortex (PFC) and hippocampus (HIP) was converted into cDNA using a high capacity RNA-to-cDNA Kit (Applied Biosystems, Foster City, CA, USA). Levels of mRNA expression were quantified using an ABI Prism 7900HT Real-Time PCR System (Applied Biosystems) with the KAPA SYBR^® FAST qPCR Kit (Kapa Biosystems, Boston, MA, USA), according to the manufacturer’s directions. The primer pairs for eight candidate markers confirmed by WB (Table 2), proinflammatory mediators (Myxovirus resistance (Mx) 2: Mx1; Interferon α: Ifna; Interferon β: Ifnb; Interferon γ: Ifng; Interleukin 1β: Il1b; Interleukin 6: Il6; Tumor necrosis factor α: Tnf; Nuclear factor of kappa light polypeptide gene enhancer in B cells 1, p105: Nfkb), and a housekeeping gene (β-actin: Actb) are described in Supplementary Table S3.

Total RNA was isolated using a NucleoSpin^® RNA kit (MACHEREY-NAGEL) following the user’s instruction. First-strand cDNAs were synthesized from RNase-free DNase I treated (Invitrogen, Waltham, MA, USA) total RNA to eliminate genomic DNA contamination using a High Capacity cDNA synthesis™ system (Applied Biosystems, Waltham, MA, USA). qRT-PCR was performed with ABI Prism 7900 HT Real-time PCR system (Applied Biosystems, Waltham, MA, USA) using Power SYBR^® Green PCR Master Mix (Applied Biosystems, Waltham, MA, USA). Three replicates were performed for the analysis of each sample. Potato 18S-rRNA as an internal control and the relative expression levels of the target gene were determined. For relative quantification, the 2^−ΔΔCT method between conditions in RT-qPCR was applied. The sequence of the primers is listed in Supplementary Table S6.