Realplex real time pcr system

The expression of select osteogenic marker genes was assessed by quantitative PCR (qPCR), essentially as previously described, with some minor modifications [35 ,67 (link)]. The Taqman primers (from Allied Biosystems, ThermoFisher) were: ALPL (Mm00475834_m1), RUNx2 (Mm00501584_m1) and Osteonectin/SPARC/BM40 (Mm00486332_m1). Quantitative PCR (qPCR) was performed in a RealPlex Real-Time PCR System (Eppendorf, Enfield, CT) with fast thermal cycling, as previously described [35 ]. The level of expression of each gene was normalized to the level of expression of a common standard housekeeping gene, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and a relative expression set against the control at 1G in MM. Fold change was calculated via the comparative CT method (2^−ΔΔCT) [68 (link)].

To evaluate the gene expression of inducible nitric oxide synthase, 2 x 10⁶ cells per well were cultured for 18 h in 6-well plates in RPMI medium (10% FCS) in the presence of different concentrations of memantine (ranging from 1 to 100 μM) or none (control) and were stimulated by 10 μg/ml LPS for 18 h. After the incubation time, the supernatant was discarded, and the adhered cells were homogenized with Trizol for RNA extraction (Thermo Fisher Scientific). cDNA was synthetized using the Reverse Transcription Kit SuperScriptII (Thermo Fisher Scientific). qPCR was performed with SYBR Green (Fermentas) for detecting the gene expression levels of iNOS. All reactions were run in triplicate on an Eppendorf RealPlex Real Time PCR System (Eppendorf) with the standard thermal cycling conditions. The runs were normalized with the ACT-β gene. The threshold cycle (2^-ΔΔCt) method of comparative PCR was used for the data analysis.

The expression of select osteogenic marker genes was determined by quantitative PCR (qPCR) essentially as previously described, with some minor modifications^{34 (link)}. In brief, total RNA was extracted from 7F2 cells using a modified version of a hybrid Tri Reagent (Sigma-Aldrich, USA)/RNEasy® protocol. RNA was quantified using a NanoDrop Spectrophotometer (Thermo Scientific, Waltham, MA), and concentrations were brought to a uniform level using additional RNase-free water. RNA was reverse transcribed to cDNA using the high-capacity cDNA reverse transcription kit (Applied Biosystems, Foster City, CA) according to the manufacturer’s instructions. The cDNA was amplified in TaqMan Fast universal PCR master mix with TaqMan assay primers and probes according to the manufacturer’s instruction. Genes of interest were: ALPL (Mm00475834_m1), RUNx2 (Mm00501584_m1) and Sparc/osteonectin/BM40 (Mm00486332_m1). Quantitative PCR (qPCR) was performed in a RealPlex Real-Time PCR System (Eppendorf, Enfield, CT) with fast thermal cycling as described by Taqman (Applied Biosystems). The level of expression of each gene was normalized to the level of expression of a common standard housekeeping gene, glyceraldehyde 3-phosphate dehydrogenase (GAPDH) to determine the fold change in up/downregulation of the genes of interest using the comparative CT method (2-ΔΔCT)^{35 (link)}.

qRT-PCR was performed to investigate the changes in relative expression levels of genes (Ces2a, Cyp1a2, Prom1, Fmo3, Alas1, Derl3). Assays were performed using the QuantiTect SYBR Green PCR kit (Qiagen, Hilden, Germany) in an Eppendorf Realplex Real-Time PCR system (Eppendorf, Hamburg, Germany). Gene expression levels were normalized to GAPDH. The experiments were performed in duplicate with liver samples prepared from 3 animals per group. The fold changes of the selected genes were analyzed by the 2^˗ΔΔCT method [41 (link)]. qRT-PCR primers are shown in Supplementary Table 3.

Total RNA from cells or tissues was isolated by using PureLink^TM RNA mini kit (Ambion). cDNA reverse transcription was performed with High Capacity cDNA Reverse Transcription kit (Applied Bio System) and the amplified product was separated by agarose gel electrophoresis. Sequences of primers used are synthesized by IDT DNA Inc. and listed in Supplementary Table 1. Quantitative RT-PCR was performed with TaqMan PCR Master Mix (Applied Bio System) on Eppendorf Realplex Real-Time PCR System. The TaqMan probes are listed in Supplementary Table 1. Data were normalized to β-actin mRNA levels.

Total RNA was isolated after 1 h upon cLIUS stimulation of MSCs in 2D. MSCs that received no cLIUS stimulation served as controls. Briefly, the medium was removed and cells were washed with HBSS (Gibco, USA). Cells were then lysed by adding 100 μl of Trizol (Invitrogen, USA) per well. RNA was then extracted from the homogenized cell lysates using the RNeasy Mini Kit (Qiagen, USA) as per manufacturer’s protocol. Cell lysates pooled from two wells served as one replicate. Three such replicates from three independent experiments were used for analysis (n = 3). qRT-PCR was carried out in Realplex™ real-time PCR system (Eppendorf, USA) using TaqMan® RNA-to-CT™ 1-Step Kit (Life Technologies, USA) as per the manufacturer’s guidelines. TaqMan® probes (Life Technologies, USA) for SOX9 (Hs00165814_m1) was used to quantify mRNA expression of SOX9. The expression of mRNA transcripts was normalized to the housekeeping gene, GAPDH (Hs02786624_g1); expression and relative expression levels were calculated using the 2^−ΔΔCt method.