Thermal cycler

All plasmid and chromosomal nucleic acid manipulations were performed by standard techniques [99 ]. Plasmid DNA was transformed into E. coli strain DH5α (Protein Express, Cincinnati, OH). To detect the presence of insert DNA, X-Gal (5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside; 40 μg/ml) was added to agar media. Restriction endonucleases, the Klenow fragment of DNA polymerase I, T4 DNA polymerase, and T4 DNA ligase were used as specified by the vendor (Invitrogen/ Gibco-BRL Corp., Gaithersburg, MD). Plasmid DNA was isolated by using plasmid miniprep isolation kits (Qiagen), and restriction fragments were recovered from agarose gels by using SeaPlaque low-melting-point agarose (FMC BioProducts, Rockland, ME). PCRs were performed by using Pfu DNA polymerase (BRL) and appropriate primers in an MJ Research thermal cycler, with 30 cycles of denaturation (2 min, 94°C), annealing (1 min, 54°C), and extension (1 min 30 s, 72°C). Amplified DNA fragments were gel purified, cloned into pCR2.1 (Invitrogen), and sequenced.

The expression levels of CREB, BDNF, IL-6 and TNF-α mRNA were measured by reverse transcription-polymerase chain reaction (RT-PCR), as described previously [30 ]. The rats were anesthetized via inhalation of isoflurane (4%) and decapitated with a guillotine; the brains were then harvested. A coronal section of the hippocampus was dissected from the brain. Total RNA was isolated from homogenates using the miRNeasy Kit (Qiagen, Hilden, Germany) according to the manufacturer’s protocol. cDNA was synthesized from total RNA using reverse transcriptase (Takara Bio, Otsu, Japan), and then amplified by PCR using Taq DNA polymerase (Takara, Kyoto, Japan) on a thermal cycler (MJ Research, Watertown, MA, USA). cDNA expression levels were eventually determined by adjusting each band intensity to that of the glyceraldehyde 3-phosphate dehydrogenase (GADPH) control.

We measured the expression levels of BDNF and tropomyosin receptor kinase B (TrkB) mRNA by reverse transcription polymerase chain reaction (RT-PCR). We rapidly removed the rats' brains and stored them at −80°C until use. Total RNA was prepared from the homogenized Hipp using 1 mL of TRIzol reagent (Invitrogen Co., Carlsbad, CA, USA). Complementary DNA was synthesized from total RNA using reverse transcriptase (Takara Co., Shiga, Japan). For polymerase chain reaction (PCR) analysis, primers specific for each mRNA sequence were used with a previously reported reagent [29 (link)]. PCR amplification was performed at 56°C for 28 cycles for BDNF and 57°C for 30 cycles for TrkB using a thermal cycler (MJ Research, Watertown, MA, USA). The PCR products were separated on a 1.2% agarose gel and stained with ethidium bromide. The density of each band was quantified using an image analysis system (i-MaxTM, CoreBio System Co., Seoul, South Korea). The expression levels were compared by calculating the relative density of each target band to the relative density of glyceraldehyde 3-phosphate dehydrogenase (GAPDH).

Total genomic DNA was extracted using either the CTAB method [27 ] as modified by [28 ] or the Nucleospin Plant (Macherey-Nagel, GmbH et Co., Düren, Germany), depending on the quality of the vegetal material. Polymerase chain reaction (PCR) was performed by using an MJ Research Inc. thermal cycler (Watertown, Massachusetts, USA) in a 25 μL volume. Direct sequencing of the amplified DNA segment was performed with the Big Dye Terminator Cycle Sequencing v3.1 (PE Biosystems, Foster City, California, USA). Nucleotide sequencing was carried out at the Serveis Científics i Tecnològics (Universitat de Barcelona) on an ABI PRISM 3700 DNA analyzer (PE Biosystems, Foster City, California, USA). ITS region—Double-stranded DNA of the ITS region (including ITS1, 5.8S gene, and ITS2) was amplified by PCR with ITS1f and ITS4r primers [29 ]. The PCR profile used for amplification was 94°C 3min; 30 × (94°C 20 s; 55°C 1 min; 72°C 1 min); 72°C 10 min. Only the ITS4 primer was used for sequencing in most cases. ETS region—Double-stranded DNA of the ETS region was amplified with the ETS1f and 18SETSr primers [30 ]. The PCR profile used for amplification was 94°C 3min; 30 × (94°C 20 s; 50°C 1 min s; 72°C 1 min); 72°C 10 min. Both ETS1f and 18SETS were used as sequencing primers, and also the internal primers AST1F and AST1R [31 ] were used occasionally.

After 2 washes with cold RH buffer, Caco-2 cells were lysed with RLT lysis buffer (Quiagen, Courtaboeuf, France). mRNA extraction was then performed using commercially available RNeasy mini kit (Qiagen, Courtaboeuf, France) in accordance with manufacturer’s instructions. The amount of RNA and the purity were measured via spectrophotometer (absorbance at A260 nm and A280 nm). Reverse transcription (RT) was performed in a final volume of 20 µL containing 250 ng of RNA, RT-MIX (BioRad iScriptTM Reverse Transcription Supermix) and H₂O. The samples were incubated in Thermal Cycler (MJ Research, Hampton, NH, USA) using the following protocol: priming 5 min at 25 °C, reverse transcription 30 min at 42 °C and heating to 85 °C for 5 min to inactivated the reverse transcriptase. Expression levels of transporters studied were evaluated by real-time PCR (qPCR). The templates obtained by the RT were used for qPCR amplifications employing CFX96 Real Time System (Biorad), the Supermix (SsoFastTM EvaGreen Supermix, BioRad) and specific primers listed in Table 2. Specificity and efficacy of each primer was tested before their use. The experimental cycling step condition consisting in an initial enzyme activation at 95 °C for 30”, 39 cycles of denaturation at 95 °C for 5” and annealing/extension step at 60 °C for 5”. β-actin was used as housekeeping gene to normalize mRNA.