Rt pcr kit

Fibroblast cultures were homogenized 24 h after treatment (FB+VIS or FB+wIRA) and mRNA was isolated by RNeasy kit (Qiagen, Hilden, Germany). mRNA concentration and its quality (A260:A280 ratio ≥ 1.8) were checked by spectrophotometry (NanoDropND1000, Nanodrop Technologies, Wilmington, DE, USA). The mRNA expression was analyzed by RT-PCR (Reverse Transcription Polymerase Chain Reaction), using a RT-PCR kit (Roche) and specific primers for MMP-1, fibrillin 1, fibrillin 2 and elastin (Table S2). Semi-quantitative analysis of RT-PCR results was performed using DDCt method (Light Cycler^® 480 Software, version 1.5, Roche Molecular Systems, Indianapolis, IN, USA), which corrects the data obtained from each sample relative to rRNA 18S reference value from each cDNA. Results were transformed into RQ (relative quantity) values using the control samples for normalization (RA = 1.00). All RT-PCR analyses were carried out at Genomics Unit, Parque Científico de Madrid, Madrid, Spain.

Total RNA was purified from different organs of Arabidopsis, including seeds at several time-points during germination (Oñate-Sánchez and Vicente-Carbajosa, 2008 (link)), and used to synthesized cDNA from 1-μg RNA samples (RT-PCR Kit from Roche Applied Science, Mannheim, Germany). The specific primers for the RT-qPCR analyses are shown in Supplementary Table S1 and the expression of the Actin 8 (ACT-8, At1g49240) gene was used to normalize the data (Graeber et al., 2011 (link)). Eco-Real-Time PCR System (Illumina, San Diego, CA, USA) was used and for each 10-μl reaction: 1 μl cDNA sample was mixed with 5 μl of FastStart Universal SYBR Green Master (Roche Applied Sciences), 0.25 μl of each primer (final concentration 500 nM), plus sterile water up to the final volume. The thermal-cycling conditions were 95 °C for 10 min, 40 cycles for 10 s at 95 °C, and 30 s at 60 °C. The melting curve was designed to increase from 55 to 95 °C and primer efficiencies were estimated from a calibration dilution curve and slope calculation (Supplementary Table S1). This analysis was performed with three different biological samples for each time point. Expression levels were determined as the number of cycles needed for the amplification to reach a threshold fixed in the exponential phase of the PCR reaction (Ct; Pfaffl, 2001 (link)).

The 6-mm coronal brain slices, 6 to 12 mm apart starting from the frontal pole of the frontal cortex, were prepared by a brain matrix device (RBM-40000, ASI Instruments, Houston, TX, USA). Total RNA was prepared using a TRIzol reagent (Thermo Fisher Scientific, Waltham, MA, USA) and 1 µg RNA samples were used for cDNA synthesis using an RT-PCR kit (Roche, Mannheim, Germany). Sequences for primers sets were summarized in Table 2.

Total RNA was prepared using TRIzol reagent (Gibco BRL, Gaithersburg, MD), and 1000 ng aliquots of RNA samples were used for cDNA synthesis, which was conducted using a RT-PCR kit (Roche, Mannheim, Germany). The sequences of the rat interleukin-1β (IL-1β), TNF-α, IL-6, and GAPDH primers used were described previously36 (link).

RNA was prepared using TRIzol reagent (Gibco BRL, Gaithersburg, MD), and 1 μg aliquots were used for cDNA synthesis, which was conducted using a RT-PCR kit (Roche, Mannheim, Germany). The sequences of the rat tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), cyclooxygenase-2 (Cox-2), inducible NO synthase (iNOS), and GAPDH primers used were described previously14 (link).

Total RNA was extracted using TRIZOL Reagent (Invitrogen), and cDNA was synthesized using a reverse transcription (RT)-PCR Kit (Roche) according to the manufacturer's instructions. Quantitative real-time PCR (qRT-PCR) was performed using the SYBR Green PCR Kit (Applied Biosystems) and an ABI PRISM 7900 Sequence Detector (Applied Biosystems). Specificity of primers was verified by dissociation curve analysis. Data was analyzed using ABI SDS v2.4 software (Applied Biosystems). All qRT-PCR reactions were performed in duplicates. Housekeeping gene GAPDH was used as an internal control. Using primers listed in Supplementary Table 5.