Peqgold rna trifast

For molecular analysis, intestinal tissue samples were snap frozen in liquid nitrogen and kept at −80 °C until further processing. After tissue collection in cryo-tubes containing 1.4 mm ceramic beads and homogenization in a FastPrep (Precelly 24, Peqlab, Erlangen, Germany) at 5000× g for 15 s, RNA was isolated by phenol-chloroform extraction using peqGold RNA TriFast (PeqLab, Erlangen, Germany), as previously described [45 (link)]. The purity of the obtained RNA was measured using 260/280 ratios of optical densities (Nanodrop 1000, PeqLab, Erlangen, Germany). Complementary DNA was obtained by reverse transcription using M-MLV transcriptase and random hexanucleotide primers (Invitrogen, Carlsbad, CA, USA). Relative mRNA expression levels were analyzed with quantitative real-time PCR (Bio-Rad, Feldkirchen, Germany). The ratio between the gene of interest (Occludin, sense: 5′-CCCAGGTGGCAGGTAGATTA, antisense: 5′-AGGCCTGTTTTGTGAGCACT; Claudin-1, sense: 5′-GTGGTGTTGGGTAAGAGGTTGT, antisense: 5′-AAAAGATGTGGATGGCTGTCA; Zonulin, sense: 5′-ACTGGGTCCAGGAAACAATG, antisense: 5′-TCCTCTTCCAGGGTGAATTG) and the reference gene (Cyclophilin A, sense: 5′-GGCAAATGCTGGACCAAACAC, antisense: 5′-TTAGAGTTGTCCACAGTCGGG AGATG) was calculated and expressed as the fold change relative to the control group.

Total RNA was extracted from testis tissue in different experimental groups using PeqGOLD RNA TriFast (PeqLab, Germany). A spectrophotometric method at 260/280 nm wavelength was used to determine total RNA amount and quality. 2 μg of total RNA was applied for complementary DNA (cDNA) synthesis in a total volume of 20 μl using RevertAid ${}^{\text{TM}}$ first strand cDNA Synthesis Kit (Aryatous, Iran). Measurement of expression levels of each gene was performed in a 20 μl mixture consisting of 2 μl of cDNA (5-fold diluted), 0.5 μl of 5 mmol/l solutions for each of the forward and reverse primers, and 10 μl of 2x SYBR green DNA PCR Master Mix (Yekta Tajhiz Azma, Iran). Primer sequences are listed in table I. Each sample was duplicated and performed on the LightCyclerⓇ 96 System (Roche, USA). The expression ratio was calculated using a relative formula based on the comparative CT method (2^-ΔΔCt) (13).

Total RNA was extracted from testis tissue in different experimental groups using PeqGOLD RNA TriFast (PeqLab, Germany). A spectrophotometric method at 260/280 nm wavelength was used to determine total RNA amount and quality. 2 μg of total RNA was applied for complementary DNA (cDNA) synthesis in a total volume of 20 μl using RevertAid ${}^{\text{TM}}$ first strand cDNA Synthesis Kit (Aryatous, Iran). Measurement of expression levels of each gene was performed in a 20 μl mixture consisting of 2 μl of cDNA (5-fold diluted), 0.5 μl of 5 mmol/l solutions for each of the forward and reverse primers, and 10 μl of 2x SYBR green DNA PCR Master Mix (Yekta Tajhiz Azma, Iran). Primer sequences are listed in table I. Each sample was duplicated and performed on the LightCyclerⓇ 96 System (Roche, USA). The expression ratio was calculated using a relative formula based on the comparative CT method (2^-ΔΔCt) (13).