Bio gel imagery apparatus

An RT-PCR analysis was performed to determine the effects of myricetin on MMP-9, BDNF and TrkB mRNA expression. Total RNA was extracted from the hippocampal tissues using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA). First-strand cDNA was synthesized using the Revert Aid First Strand cDNA Synthesis kit (Fermentas; Thermo Fisher Scientific, Inc., Waltham, MA, USA). PCR reactions were performed using the 7300 Real-Time PCR system (Applied Biosystems; Thermo Fisher Scientific, Inc., Waltham, MA, USA). The following primers were used: Forward, 5′-CGAAGAGCTGCTGGATGAG-3′, and reverse, 5′-ATGGGATTACACTTGGTCTCG-3′, for BDNF; forward, 5′-CCTCCACGGATGTTGCTGA-3′, and reverse, 5′-GGCTGTTGGTGATACCGAAGTA-3′ for TrkB; forward, 5′-GTCTTCCCCTTCGTCTTCCT-3′, and reverse, 5′-GCTGGATGCCTTTTATGTCG-3′ for MMP-9; forward, 5′-CCGTATCGGACGCCTGGTTA-3′, and reverse, 5′-GGCTGTTGGTGATACCGAAGTA-3′, for GAPDH. GAPDH was used as the internal control. PCR products were separated by 1% agarose gel electrophoresis and visualized using ethidium bromide (0.05%) staining. The band intensities of the products were analyzed with the Bio-Gel imagery apparatus with Quantity One^® 1-D analysis software (4.3.0; Bio-Rad Laboratories, Inc., Hercules, CA, USA).

RT-PCR was performed to assess the influence of genistein on BDNF and TrkB gene expression in the hippocampal tissues of isoflurane anesthesia-treated rat pups. Total RNA was isolated from the hippocampi using Trizol (Invitrogen, Carlsbad, CA, USA), and the RNA concentration was determined using a Nanodrop spectrophotometer (ND 1000; Bio-Rad, Hercules, CA, USA). First-strand complementary DNA (cDNA) was synthesized using the Revert Aid First Strand cDNA Synthesis Kit (Fermentas, Glen Burnie, MD, USA). PCR was performed according to the manufacturer's protocol. The primer sequences for BDNF and TrkB were as follows: BDNF, Forward: 5'-CGAAGAGCTGCTGGATGAG-3', Reverse: 5'-ATGGGATTACACTTGGTCTCG-3'. TrkB, Forward: 5'-CCTCCACGGATGTTGCTGA-3', Reverse: 5'-GGCTGTTGGTGATACCGAAGTA-3'. GAPDH expression was assessed as an internal control using the following primer sequences: Forward: 5'-CCGTATCGGACGCCTGGTTA-3', Reverse: 5'-GGCTGTTGGTGATACCGAAGTA-3'. PCR products were separated on agarose gels (1%) and stained with 0.05% ethidium bromide. Band intensities were analyzed using a Bio Gel imagery apparatus (Bio Rad).

RT-PCR analysis was done to explore the influence of 6-shogaol on the levels of VEGF and Flk-1 m-RNA by TaqMan RT-PCR. The endometrial tissue total RNA was extracted as per manufacturer's protocol using TRIZOL reagent (Invitrogen, Carlsbad, CA, USA). Total isolated RNA (2 µg) was used as a template for synthesising cDNA and by using the SuperScript II reverse transcriptase kit (Invitrogen) the first strand was synthesized. With SYBR green fluorescence PCR was carried out using 7300 Real-Time PCR System (Applied Biosystems). The following primers were used for amplification - Flk-1-sense: 5′-GCACTGAATTATGGGAGA-3′, antisense-5′-ATGTGATTTTCTTCTTGATG-3′; VEGF-sense: 5′-ACCATGAACTTTCTGCTC-3′, antisense-5′-GGACGGCTTGAAGATATA-3′; GAPDH-sense: 5′-CACCACCATGGAGAAGGC-3′, antisense: 5′-CCATCCACAGTCTTCTGA-3′. The PCR products were then separated on agarose gel electrophoresis (2%). The bands were stained with ethidium bromide (0.05%) and the intensities were analyzed by Bio-Gel imagery apparatus (Bio-Rad, USA).