gcl catalytic subunit  (Novus Biologicals)

Journal: PLoS ONE

Article Title: Probiotics Prevent Intestinal Barrier Dysfunction in Acute Pancreatitis in Rats via Induction of Ileal Mucosal Glutathione Biosynthesis

doi: 10.1371/journal.pone.0004512

Figure Lengend Snippet: After 5 days of pre-treatment with placebo (pla, n = 12) or probiotics (pro, n = 12), rats were subjected to acute pancreatitis (AP, n = 12), a sham-procedure (n = 12) or not operated (control, n = 5). Six hours after induction of the AP or sham-procedure, tissue cysteine availability (A) and mucosal glutamate-cysteine-ligase (GCL) activity (B) were determined in ileum samples. (C) RT-PCR was conducted on ileal mRNA. PCR products of specific primers for the catalytic (GCLc, 65 bp) and the modulatory (GCLm, 81 bp) subunit of GCL and for 18S rRNA (65 bp) as control were identified on 2.5% agarose gel, using a GeneRuler 50 bp DNA Ladder (Fermentas GMBH, St. Leon-Rot, Germany). mRNA expression of (D) GCLc and (E) GCLm were quantified. Data are normalized to 18S rRNA expression and expressed as ratio to control animals. The graphs show average (±SD). All analyses were run in triplicates. Comparisons were performed using ANOVA followed by Tukey's HSD.

Article Snippet: RT-PCR with mRNA-specific primers for the catalytic (GCLC) and modifier (GCLM) subunits of GCL and 18S rRNA as a reference gene was performed (GCLC-forward 5′-ggcgatgttcttgaaactctg-3′ , GCLC-reverse 5′-cagagggttgggtggttg-3′ ; GCLM-forward 5′-ctgactcacaatgacccaaaag-3′ , GCLM-reverse 5′-ttcaatgtcagggatgctttc-3′ ; 18S rRNA-forward 5′-aatcagttatggttcctttgtcg-3′ , 18S rRNA-reverse 5′-gctctagaattaccacagttatccaa-3′ ; Sigma-Aldrich) and mRNA levels were quantified using SYBR Green based detection.

Techniques: Activity Assay, Reverse Transcription Polymerase Chain Reaction, Agarose Gel Electrophoresis, Expressing