Data about methods of RNA isolation and cDNA synthesis were described in our previous research (Gavrilović et al., 2012 (link)). Specifically, prefrontal RNAs were isolated by using TRIZOL reagent (Invitrogen, Waltham, MA, USA). As stated in the manufacturer’s protocol, reverse transcription was carried out using Ready-To-Go You-Prime First-Strand Bead (Amersham Biosciences, Amersham, UK) and pd (N)6 Random Hexamer (Amersham Biosciences, Amersham, UK) primer. The more detailed description of the procedure was provided in our previous protocol (Gavrilović et al., 2012 (link)). Gene expression of antioxidant enzymes was performed using quantitative real-time RT-PCR method. Determination of SOD1, SOD2, CAT and GPx mRNA was performed by TaqMan PCR assays (Applied Biosystems, Waltham, MA, USA) for SOD1 (Rn00566938_m1), SOD2 (Rn00690587_g1), CAT (Rn00560930_m1) and GPx (Rn00577994_g1). The endogenous control was included in each analysis to correct for the differences in the inter-assay amplification efficiency and all transcripts were normalised to cyclophyline A (Rn00690933_m1) expression (Gavrilović et al., 2012 (link)). The relative expression of antioxidant enzymes was normalized to cyclophyline A and expressed in relation to the calibrator, i.e., the control sample, following our previous protocol (Gavrilović et al., 2012 (link)).
Pd n 6 random hexamer
Manufactured by Cytiva
Sourced in United Kingdom
Pd (N)6 Random Hexamer is a reagent used in molecular biology applications. It serves as a primer for reverse transcription, facilitating the synthesis of complementary DNA (cDNA) from RNA templates. The product consists of a mixture of short, randomly generated DNA sequences that can anneal to various RNA targets, enabling the conversion of RNA to cDNA for further analysis or downstream applications.
Automatically generated - may contain errors
2 protocols using pd n 6 random hexamer
1
Quantifying Antioxidant Enzyme Expression
2
Quantifying Hepatic Cytokine Expression
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Frozen hepatic sample were used to quantify the genic expression of TNFα, IL12 and IFNγ. The tissue fragments were homogenized and submitted for analysis of the expression of total RNA using a specific kit (RNeasy Mini Kit, QIAGEN, CA, USA). During the procedure, complementary DNA (cDNA) was acquired by reverse transcription, beginning with 5 μg of total RNA, using a retrotranscription kit (Ready-To-Go You-Prime First-Strand Beads and pd(N)6 Random Hexamer as primers (Amersham Biosciences). mRNA amplification, with concurrent quantification, was conducted by RT-PCR StepOnePlusTM (Applied Biosystems Inc., Foster City, CA, USA) using specific primers to TNFα, IL12, IFNγ and 18S RNA transcripts (Assays-on-Demand Gene Expression Products, Applied Biosystems) and a specific Taq Polymerase enzyme (TaqMan Universal PCR Master Mix, No AmpErase UNG -2X, Applied Biosystems). All experiments were performed in duplicate and the results were normalized to 18S rRNA expression.
Statistical analysis. Data were analyzed using the GraphPad Prism software 4.0 (GraphPad Software, San Diego, CA, USA). All data are reported as mean±standard deviation (SD). Statistical comparisons of the groups were performed by non-parametric Kruskal-Wallis one-way analysis of variance followed by Dunn's posttest or Mann-Whitney test. Probability value p>0.05 was considered to be statistically significant.
Statistical analysis. Data were analyzed using the GraphPad Prism software 4.0 (GraphPad Software, San Diego, CA, USA). All data are reported as mean±standard deviation (SD). Statistical comparisons of the groups were performed by non-parametric Kruskal-Wallis one-way analysis of variance followed by Dunn's posttest or Mann-Whitney test. Probability value p>0.05 was considered to be statistically significant.
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