Rotor gene sybr green

MCF-7 and MCF10A cells were treated with cortisol for 30 minutes and for 24 h (1 μM). For RNA extraction from tumours, 30 mg of tissue per sample was homogenized. RNA was extracted using an RNeasy Kit (Qiagen, UK) and cDNA was synthesised using a Quantitect Reverse Transcription kit (Qiagen, UK) as per the manufacturer’s instructions. A Rotor-Gene SYBR Green (Qiagen, UK) master mix was prepared according to the manufacturer’s instructions using Quantitect Primer Assay for human NOS2 (Qiagen, UK) or mouse NOS2. The sense and antisense primers for mouse NOS2 were 5′-AATGGCAACATCAGGTCGGCCATCACT-3′ and 5′-GCTGTGTGTCACAGAAGTCTCGAACTC-3′ respectively (Eurofins). cDNA was analysed in the Rotor-Gene qRT-PCR thermocycler and presented as fold change in expression normalised against β-actin.

Sodium azide (0.5%; Sigma) was added to exponentially growing cells to prevent further metabolism. Chromosomal DNA was isolated using a DNeasy Blood and Tissue Kit (Qiagen). The DNA replication origin (oriC) region was amplified using primers 5′-GAATTCCTTCAGGCCATTGA-3′ and 5′-GATTTCTGGCGAATTGGAAG-3′; the DNA replication terminus (ter) region was amplified using primers 5′-TCCATATCCTCGCTCCTACG-3′ and 5′-ATTCTGCTGATGTGCAATGG-3′. Either Rotor-Gene SYBR Green (Qiagen) or GoTaq (Promega) qPCR mix was used for PCR reactions. Q-PCR was performed in a Rotor-Gene Q Instrument (Qiagen). By use of crossing points (C_T) and PCR efficiency a relative quantification analysis (ΔΔC_T) was performed using Rotor-Gene Software version 2.0.2 (Qiagen) to determine the origin:terminus (ori:ter) ratio of each sample. These results were normalized to the ori:ter ratio of a DNA sample from B. subtilis spores which only contain one chromosome and thus have an ori/ter ratio of 1.

Total RNA was extracted from complete seedlings of the different ages using the innuPREP Plant RNA kit (Analytik Jena Bio solutions). cDNA synthesis was carried out by using the QuantiTect Reverse Transcription Kit (QIAGEN). Rotor-Gene SYBR Green (QIAGEN) and the Rotor-gene-Q cycler (QIAGEN) were used for performing the quantitative PCRs. Generated data were quality-controlled and normalized to the reference genes FASS (AT5G18580) and SAND (AT2G28390) using the qbasePLUS software (Hellemans et al, 2007 (link)). For validation of intron retention events visualized on the Integrative Genomics Viewer software, two primers pairs were designed to specifically detect intron and exon transcripts. Exon primers annealed to the exon junction, whereas intron-specific primers annealed inside the retained intron. Intron retention was quantified by dividing intron expression by exon-specific expression. All primers are listed on Table S16.

nuc-SG primers used in the secondary detection were the same as in the primary detection using SybrGreen (Table 1). Primer sequences used for mecA amplification have been previously described [14 (link)].
Amplification of nuc and mecA was performed in parallel in the PCR instrument, but in separate reactions. The reaction mix of 20 μL contained 2x RotorGene SybrGreen (Qiagen), 0,5 μM of primers (Eurogentec), directed at nuc and mecA, and 5 μL of DNA template. A real-time PCR was performed in RotorGene 3000 (Qiagen) starting with a pre-incubation step at 95°C for 5 min, followed by 35 cycles of 95°C for 5 s and 60°C for 15 s. The amplification was followed by a melting curve analysis with a ramp from 60°C to 95°C.

The total RNA was isolated using TRIzol^® reagent (Invitrogen, Waltham, MA, USA) according to the manufacturer’s instructions. The total RNA (2 µg) was reverse transcribed using 200 units of M-MLV reverse transcriptase (Invitrogen) in a 20 µL reaction mixture at 42 °C for 1 h. For quantitative real-time PCR analyses, the resulting cDNA (1 µL) was amplified in 10 µL of‘ Rotor-gene SYBR^® green (Qiagen, Hilden, Germany). The comparative cycle threshold (C_T) method was used, and β-actin served as an endogenous control. The primers are listed in the supplementary table S1.

For the extraction of RNA, cells were incubated with RNAlater (Ambion) for 1 min, washed with water and lysed in QIAzol (Qiagen). RNA was extracted according to manufacturer's instructions. 1 μg of total RNA was used to synthesize cDNA with Superscript II Reverse Transcriptase (Invitrogen LT) and oligo(dT) primers (Invitrogen LT) according to the recommended protocol.
Semi-quantitative RT-PCR was performed with GoTaq^TM DNA polymerase (Promega) according to the instructions of the producer. For quantitative real-time RT-PCR a Rotor-Gene®Q LightCycler (Qiagen) detecting Rotor Gene SYBR Green (Qiagen) was used. All values were normalized to β-actin mRNA as internal standard. Oligonucleotide primers and annealing temperatures used for gene amplification are listed in Supplementary Table S2.