Journal: Journal of Biomolecular Techniques : JBT

Article Title: RNA Purity, Real-Time PCR Sensitivity, and Colon Segment Influence mRNA Relative Expression in Murine Dextran Sodium Sulfate Experimental Colitis

doi: 10.7171/jbt.18-2903-001

Figure Lengend Snippet: qRT-PCR curves for β-actin, Eef2, and IL-6 using the 1-step protocol. Real-time fluorescence using 1:10-fold dilutions starting at 200 ng total RNA from colon run in triplicate using primers/probe for β-actin (A), Eef2 (B), and IL-6 (C). B) The standard curves generated from the Ct values of each of the dilutions and the equation of the line of best fit for β-actin (D), Eef2 (E), and IL-6 (F).

Article Snippet: For both assays, a Taqman gene-expression assay was performed using commercially available, predesigned primers and probes (Thermo Fisher Scientific) for β-actin (Mm00607939_s1), both primers and probe map within a single exon: exon 6, GenBank {"type":"entrez-nucleotide","attrs":{"text":"AK078935.1","term_id":"26098178","term_text":"AK078935.1"}} AK078935.1 : location 1233, amplicon length 115 (a qPCR without the RT step was performed to confirm that genomic DNA is not amplified); eukaryotic translation elongation factor 2 (Eef2; Mm01171435_gH), probe spans exons: exon boundary 2–3, RefSeq {"type":"entrez-nucleotide","attrs":{"text":"NM_007907.2","term_id":"237858599","term_text":"NM_007907.2"}} NM_007907.2 : location 316, amplicon length 62 bp; and IL-6 (Mm00446190_m1), probe spans exons: exon boundary 2–3, RefSeq {"type":"entrez-nucleotide","attrs":{"text":"NM_031168.1","term_id":"13624310","term_text":"NM_031168.1"}} NM_031168.1 , location 237, amplicon length 78 bp.

Techniques: Quantitative RT-PCR, Fluorescence, Generated

Journal: Journal of Biomolecular Techniques : JBT

Article Title: RNA Purity, Real-Time PCR Sensitivity, and Colon Segment Influence mRNA Relative Expression in Murine Dextran Sodium Sulfate Experimental Colitis

doi: 10.7171/jbt.18-2903-001

Figure Lengend Snippet: Variability of the proinflammatory cytokine IL-6 relative expression in different colon segments. mRNA relative expression of IL-6 in the 3 segments of the colon, proximal (A, D), middle (B, E), and distal (C, F). qRT-PCR was performed by the 1-step protocol, and data are expressed as the relative expression calculated using the Pfaffl method16 and β-actin (A–C) or Eef2 (D–F) as reference genes. Results are expressed as means ± sd; n = 5 mice per group. Mann-Whitney, *P < 0.05, **P < 0.01.

Article Snippet: For both assays, a Taqman gene-expression assay was performed using commercially available, predesigned primers and probes (Thermo Fisher Scientific) for β-actin (Mm00607939_s1), both primers and probe map within a single exon: exon 6, GenBank {"type":"entrez-nucleotide","attrs":{"text":"AK078935.1","term_id":"26098178","term_text":"AK078935.1"}} AK078935.1 : location 1233, amplicon length 115 (a qPCR without the RT step was performed to confirm that genomic DNA is not amplified); eukaryotic translation elongation factor 2 (Eef2; Mm01171435_gH), probe spans exons: exon boundary 2–3, RefSeq {"type":"entrez-nucleotide","attrs":{"text":"NM_007907.2","term_id":"237858599","term_text":"NM_007907.2"}} NM_007907.2 : location 316, amplicon length 62 bp; and IL-6 (Mm00446190_m1), probe spans exons: exon boundary 2–3, RefSeq {"type":"entrez-nucleotide","attrs":{"text":"NM_031168.1","term_id":"13624310","term_text":"NM_031168.1"}} NM_031168.1 , location 237, amplicon length 78 bp.

Techniques: Expressing, Quantitative RT-PCR, MANN-WHITNEY