Rt pcr kit

Total RNA was extracted from each cell culture flask using the RNeasy extraction kit (RNeasy micro kit, cat. no. 74004). Up to 1 × 10⁶ cells, depending on the cell line, were disrupted in buffer RLT and homogenized and disrupted. Ethanol was then added to the lysate, creating conditions that promote the selective binding of RNA to the RNeasy membrane. The samples were then applied to the RNeasy Mini spin column. Total RNA binded to the membrane, contaminants were efficiently washed away and high-quality RNA was eluted in RNase-free water. All bind, wash and elution steps were performed by centrifugation in a micro-centrifuge, with DNAse I treatment. The amount of extracted RNA was quantified by estimating the absorbance at 260 nm. The purity of the RNA was checked by measuring the ratio of the absorbance at 260 and 280 nm, where a ratio ranging from 1.8 to 2.0 was taken to be pure. The absence of degradation of the RNA was verified by RNA electrophoresis on a 1.5% agarose gel containing ethidium bromide. First-strand cDNA was generated from 1 μg of each flask using the High-Capacity cDNA Archive Kit, Model for One-Step RT-PCR procedures (RT-PCR kit- BioRad-USA, cat. no. 345-0412), according to the manufacturer’s protocol.

qRT-PCR was carried out as described previously (20 (link)) in a CFX98 multicolor system (Bio-Rad) using 2 μg of total RNA reverse transcribed with an RT-PCR kit (Bio-Rad). Single-product amplification was confirmed by melting-curve analysis, and primer efficiency was near 100% in all experiments. Quantifications are expressed in arbitrary units, and target mRNA abundance was normalized to the expression of GAPDH by the method of Pfaffl (52 (link)). All the qRT-PCR results are representative of at least three independent experiments (n > 3). A list of primers is provided in the supplemental material.

Total RNA was extracted from the testis of rabbit, and reverse transcribed to cDNA using Bio-Rad RT-PCR kit. PCR was performed in a 25 μL reaction volume using Bio-Rad IQ5 thermometer. Primers were initially selected based on homology to human and then extended using primer walking to obtain a full length coding sequence which was cloned into the pcDNA3.1 directional topo expression vector kit. Partial and full length clones were sent to Genewiz (South Plainfield, NJ) for sequencing and the resulting sequences aligned. ExPASy (http://ca.expasy.org/tools/dna.html), Prosite (http://prosite.expasy.org), and CDD algorithms (http://www.ncbi.nlm.nih.gov/Structure/cdd/cdd.shtml) were used to translate as well as to detect the presence of functional motifs and conserved regions in the protein. Predicted rabbit Nox5, human Nox5 as well as Nox5 sequences from other species were downloaded from the National Center for Biotechnology Information (NCBI) (http://ncbi.nlm.nih.gov) and Genome Browser (http://genome.ucsc.edu) from University of California Santa Cruz (UCSC). Bioedit software was utilized for DNA and protein sequences alignment and Mega 4 software ver. 4.0 was used for illustrating dendrogram by the neighbor-joining method (Tamura et al., 2007 (link)).

cDNA synthesis of mRNA samples was processed according to the Bio-Rad RT-PCR kit. Quantitative PCR was performed using the iQSYBR Green master-mix kit (Biorad, Hercules, CA, USA) according to manufacturer’s instructions. qPCR amplification and detection were performed on the RT-PCR cycler (ROTOR GENE RG-3000 from Corbett research). PGK1 housekeeping gene was used as a control. Data were analyzed using Rotor-Gene Real-Time Analysis Software 6.0.14.

A 3 mL overnight bacterial culture was centrifuged, and bacterial RNA was extracted using a PureLink RNA Mini Kit (Invitrogen) following the manufacturer’s instructions. RNA was reverse-transcribed using a reverse transcription kit (TaKaRa) to obtain cDNA and RT-PCR was performed using an RT-PCR kit (Bio-Rad). Expression levels of wzb,wzc and wzi were determined based on the results of RT-qPCR. 16sRNA was used as an internal reference . The control strain was AB ATCC17978. The primers are listed in Supplementary Table S2.

Total RNA was extracted using a Qiagen, Hilden, Germany RNA kit (Qiagen, Hilden, Germany). Samples were treated with DNase (Sigma-Aldrich, St. Louis, MO, USA) to exclude DNA contamination. The concentration and quality of RNA were determined according to optical density (OD) measurement. mRNA was reverse transcribed into cDNA using an RT-PCR kit (Bio-Rad, Hercules, CA, USA). Advanced Universal SYBR Green Supermix was used for quantitative RT-PCR in a CFX96 real-time thermocycler (Bio-Rad, Hercules, CA, USA) to determine gene expression levels of UCP1, PGC-1a, PRDM16, FABP4, CIDEA, PPAR-γ. The primer sequences are listed in Table 1. Fold changes in gene expression was normalized to β-actin expression and were plotted and compared between groups.