Rt2 real time sybr green rox pcr master mix

To determine the level of gene expression, total RNA was isolated using QIAzol lysis reagent (Qiagen Sciences LLC, Germantown, MD 20874, USA), followed by a reverse transcription reaction using QuantiTect Reverse Transcription Kit (Qiagen Sciences LLC, MD 20874, USA) according to the manufacturer’s instructions. The obtained cDNA was used for quantitative PCR using a commercial RT²—Real Time SYBR Green/ROX PCR master mix (Qiagen Sciences LLC, MD 20874, USA) and a set of lyophilized primers (Mmp9, Spp1, Cxcl12, Ppar γ, Runx2, RANKL, OPG, Ibsp, BMP10, Cxcr4, Sost, Alpl) QuantiTect Primer Assays (Qiagen Sciences LLC, MD 20874, USA). Normalization was performed according to the expression level of the reference gene Rplp1 (Qiagen Sciences LLC, MD 20874, USA). The expression level was assessed using the 2^−∆∆Ct method according to the manufacturer recommendation (Qiagen Sciences LLC, MD 20874, USA).

RNA was extracted from liver tissue using TRIzol reagent (Life Technologies), and was reverse-transcribed with a High Capacity cDNA reverse transcription kit (Applied Biosciences). qPCR was performed on a Rotor-Gene™ 3000 (Qiagen) using RT2 Real-Time SYBR^® Green/ROX PCR Master Mix (Qiagen) as per the manufacturer’s instructions, using the following themocycler conditions: 95 °C for 10 min, followed by 40 cycles of 95 °C for 15 s, 60 °C for 30 s, 72 °C for 30 s with the primers listed in Supplementary Table 1. Expression was normalized to TBP by the 2^−ΔΔCt method using Rotor-Gene™ 6000 Software (v1.7, Qiagen) and Microsoft Excel software.

The Rat Lipoprotein Signaling and Cholesterol Metabolism RT²- Profiler PCR array (Qiagen, Cat. No. PARN-080Z) was used to determine the effects of maternal LPS exposure during pregnancy on the expression of 84 genes related to lipoprotein transport and cholesterol metabolism. Briefly, the total RNA was isolated from the livers of the 8-week-old offspring rats fed a normal diet from the maternal control and LPS exposure groups, and then cDNA was prepared by using a real-time RT-PCR array first strand kit. A total volume of 25 μl of the PCR mixture, which included 12.5 μl of RT² Real-Time SYBR Green/ROX PCR master mix from Qiagen, 11.5 μl of double-distilled water, and 1 μl of template cDNA was loaded in each well of the PCR array. PCR cycles were performed according to the manufacturer's protocols. Data were analyzed using the comparative Ct method with normalization of the raw data to housekeeping genes including β-glucuronidase, hypoxanthine phosphoribosyl transferase 1, heat shock protein β-1, GAPDH, and β-actin. Controls including rat genomic DNA contamination, reverse transcription and positive PCR were performed simultaneously.

SABioscience mouse chemokine & receptors PCR array (cat. no. PAMM-022 and PAMM-011; QIAGEN, Inc, Valencia, CA) were used to compare the relative levels of cDNA between interstitial CD45^- cells or CD45+ cells from interstitium and BALF which were isolated from saline or i.n. LPS challenged (20 hrs) α7^G or α7^E260A:G mice mRNA was extracted using Qiazol (QIAGEN, Inc, Valencia, CA). cDNA was prepared using the RT2 real-time SYBR Green/Rox PCR master mix and RT2 Profiler PCR array (QIAGEN Inc.). All samples were amplified using Applied Biosystems 7900HT 384 well sequence detecting system (Life Technologies, Grand Island, NY). Results of PCR array were analyzed using the web-based RT2Profiler PCR Array Data Analysis software (version 3.5, QIAGEN).

mRNA were extracted using Nucleospin RNA kit (Macherey-Nagel). 0.5 µg of total RNA were reverse transcribed using MMLV reverse transcriptase (Invitrogen). PCR reactions were performed using QuantiTect Primer Assays (Qiagen) and RT² Real-time SYBR-Green/ROX PCR mastermix (Qiagen) and carried out using QuantStudio™ Real-Time PCR system 3 (ThermoFisher). RPLP0 gene expression was used as internal standard.