Quantifast sybr green pcr kit

Macrophage RNA was isolated using TriFast™ reagent according to the manufacturer's protocol (Peqlab, Erlangen, Germany). One μg of total RNA was reverse transcribed using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Carlsbad, CA). Quantitative real-time PCR (qPCR) was performed on a Roche LightCycler 480 (Roche Diagnostics, Palo Alto, CA) using the QuantifastTM SYBR® Green PCR kit (Qiagen, Hilden, Germany). mRNA levels were analyzed in duplicate and normalized to those of the reference gene hypoxanthine phosphoribosyltransferase (Hprt). Fold-changes and associated statistical parameters were determined by the 2^-ΔΔCT method. The following primer sequences were used:
Arg1: Fwd 5′-TGGCTTGCGAGACGTAGAC-3′; Rev 5′-GCTCAGGTGAATCGGCCTTTT-3′
Ccl5: Fwd 5′-GCTGCTTTGCCTACCTCTCC-3′; Rev 5′-TCGAGTGACAAACACGACTGC-3′
Gro1: Fwd 5′-CTGGGATTCACCTCAAGAACATC-3′; Rev 5′-CAGGGTCAAGGCAAGCCTC-3′
Hprt: Fwd 5′-TCAGTCAACGGGGGACATAAA-3′; Rev 5′-GGGGCTGTACTGCTTAACCAG-3′
Nos2: Fwd 5′-GTTCTCAGCCCAACAATACAAGA-3′; Rev 5′-GTGGACGGGTCGATGTCAC-3′
Itgax: Fwd 5′-CTGGATAGCCTTTCTTCTGCTG-3′; Rev 5′-GCACACTGTGTCCGAACTCA-3′
Mcp1: Fwd 5′-ACTGAAGCCAGCTCTCTCTTCCTC-3′; Rev 5′-TTCCTTCTTGGGGTCAGCACA-3′
Mrc1: Fwd 5′-GCTGAATCCCAGAAATTCCGC-3′; Rev 5′-ATCACAGGCATACAGGGTGAC-3′
Tnfa: Fwd 5′-CCCTCACACTCAGATCATCTTCT-3′; Rev 5′-GCTACGACGTGGGCTACAG-3′

To detect levels of mRNA expression, total RNA from mouse lungs and A549 cells was extracted using QIAGEN RNeasy Mini Kit (QIAGEN, Hilden, Germany). Extrated RNA was reverse transcribed into cDNA using QuantiTect Rev. Transcription Kit (QIAGEN, Hilden, Germany) according to the manufacturer’s instructions. Quantitative real-time PCR was performed using the QuantiFastTM SYBR Green PCR Kit (QIAGEN, Hilden, Germany) with an ABI 7500 instrument (Applied Biosystems, CA, United States). The primer sequences were available on request and were listed in Table 1. All reactions were carried out in triplicate in the same plate. The relative gene expression levels were determined by normalizing the Ct levels of the mRNA of interest to endogenous GAPDH. The ΔΔCt method was applied to evaluate and compare differential gene expression between samples. The data were analyzed using the ABI 7500 software v2.0.5 (Applied Biosystems, CA, United States).

Cells were incubated at a density of 1 × 10⁵ cells per well. Following overnight serum starvation, cells were treated with the indicated concentrations of LPA for indicated time periods. Total RNA was extracted using the RNeasy Mini kit (QIAGEN, Hilden, Germany) according to manufacturer’s protocol and quantified using NanoDrop (Thermo Fisher Scientific, Waltham, MA, USA). 1 µg of RNA was reverse-transcribed by using SuperScript^® III reverse transcription kit (Invitrogen, Waltham, MA, USA). Quantitative real-time PCR (qPCR) was performed on Applied Biosystems 7900HT Fast Real-Time PCR System using QuantifastTM SYBR^® Green PCR kit (QIAGEN, Hilden, Germany. Relative gene expression levels were normalized to hypoxanthineguanine phosphoribosyltransferase (HPRT) and calculated using ΔΔCT method [85 (link)]. Primer sequences are listed in Table 1.

Tissue samples from selected homozygous T₄ lines were snap-frozen in liquid nitrogen and stored at −80 °C until further analysis. Total RNA was extracted using the RNeasy Plant MiniKit (Qiagen, Valencia, CA, USA), as per the manufacturer’s instructions. Two micrograms of DNA-free total RNA were used for cDNA preparation. Reverse transcription reactions were performed using the Transcriptor First-Strand cDNA Synthesis Kit RT-PCR (Roche, USA) according to the manufacturer’s instructions. Primers used in the expression study are noted in Supplementary Table S2. Quantitative real-time PCR analysis was performed by following SYBR Green (QuantiFast^TM SYBR Green PCR kit, QIAGEN) chemistry using the ABI PRISM 7500 Fast Realtime Platform (Applied Biosystems). Target genes were amplified by a two-step PCR reaction with an initial denaturation at 95 °C for 5min followed by 40 cycles of 95 °C for 30s and annealing/extension at 60 °C for 30s. Relative quantification for fold-changes were calculated and Ct values were normalized against wheat ARF (ADP-ribosylation factor, AB050957.1) as defined previously by Bhati et al. (2014) (link) and Alok et al. (2015) using the 2^–ΔΔCT method (Livak and Schmitteng, 2001 (link)). The specificity of the amplification was also assessed for each gene by dissociation curve analysis. A unique peak on the dissociation curve was confirmed for each gene.

Total RNA from the brain was extracted using the RNeasy lipid tissue mini kit (QIAGEN, Hilden, Germany) according to manufacturer’s protocol and quantified using NanoDrop (Thermo Fisher Scientific, Waltham, MA, USA). RNA was reverse-transcribed by using SuperScript^® III reverse transcription kit (Invitrogen, Waltham, MA, USA). Quantitative real-time PCR (qPCR) was performed on Applied Biosystems 7900HT fast real-time PCR system using QuantifastTM SYBR^® Green PCR kit (QIAGEN, Hilden, Germany). Relative gene expression levels were normalized to hypoxanthineguanine phosphoribosyltransferase (HPRT) and calculated using ΔΔCT method [63 (link)]. Primer sequences are listed in Table 2.