Affymetrix GeneChip Human Genome HG-U133A [HG-U133A]

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Intestinal perfusion of a 40-cm segment of the small intestine in 8 healthy volunteers. 2 test days, overnight fast. Gastroduodenoscopy for tissue sampling and positioning of perfusion catheter. Continuous injection of 0.055 M glutamine (10 ml/min) in saline at 10 cm distally from the pylorus for 4 h or continuous injection of 0.055 M glucose in saline. After the injection a second gastroduodenoscopy takes place for tissue sampling. In total we have 4 samples per individual (placebo-before; placebo-after; glutamine-before; and glutamine-after injection.

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Transcription profiling by array of human duodenal mucosa after treatment with glutamine or glucose

Transcription profiling of human duodenal mucosa from healthy volunteers perfused with 0.055 M glutamine (10 ml/min) in saline at 10 cm distally from the pylorus for 4 h or continuous injection of 0.055 M glucose in saline

Intestinal perfusion of a 40-cm segment of the small intestine in 8 healthy volunteers. 2 test days, overnight fast. Gastroduodenoscopy for tissue sampling and positioning of perfusion catheter. Continuous injection of 0.055 M glutamine (10 ml/min) in saline at 10 cm distally from the pylorus for 4 h or continuous injection of 0.055 M glucose in saline. After the injection a second gastroduodenoscopy takes place for tissue sampling. In total we have 4 samples per individual (placebo-before; placebo-after; glutamine-before; and glutamine-after injection.


P-AFFY-6

Title: Affymetrix CEL analysis. Description:

This study was designed to conform to a randomized cross-over design. Each subject was tested on two separate days. Baseline duodenal biopsies, taken by standard forceps in the proximal horizontal part of the duodenum (10 cm distally from the pylorus), were obtained by gastroduodenoscopy. Biopsies were imbedded in tissue-tek (Electron Microscopy Sciences, Washington, PA, USA), snap-frozen in melting isopentane and liquid nitrogen respectively, and stored at -80ºC until analysis.

This study was designed to conform to a randomized cross-over design. Each subject was tested on two separate days. After obtaining the baseline (before) mucosal samples, a guide wire was inserted with the distal tip positioned about 10 cm distally from the pylorus, facilitated by the duodenoscopy. After retraction of the endoscope, a double lumen perfusion catheter (home made from PVC tubing, inner diameter 2.5 mm, outer diameter 4 mm) was inserted over the guide wire into the proximal small intestine with the tip of one lumen, which served as an injection port, located in the proximal descending duodenum and the tip of the second lumen, with three small holes in the last 3 cm to serve as a sample port, 40 cm distally from the injection port. After positioning, the guide wire was removed. The whole procedure was performed under short interval fluoroscopic control. To check positioning of the injection port in the duodenum throughout the experiment, intra-luminal pH was continuously measured at 5 cm proximal of the injection port by a pH electrode (Single Crystal Antimony Multi-Use pH Catheter, Synectics Medical, Queluz, Portugal). The 40-cm segment of the proximal small intestine was perfused with saline at 10 ml/min using a peristaltic pump for 195 min to obtain steady state conditions. After reaching steady state, 0.055M glucose in saline (10 ml/min) was perfused for 4 hours. All ingoing solutions were kept on 37 degrees C in a water bath. Fifteen minutes after the perfusion experiment, the perfusion catheter was removed and a gastroduodenoscopy was performed to obtain tissue samples from the same intestinal region after the perfusion. Biopsies were imbedded in tissue-tek (Electron Microscopy Sciences, Washington, PA, USA), snap-frozen in melting isopentane and liquid nitrogen respectively, and stored at -80ºC until analysis.

This study was designed to conform to a randomized cross-over design. Each subject was tested on two separate days. After obtaining the baseline (before) mucosal samples, a guide wire was inserted with the distal tip positioned about 10 cm distally from the pylorus, facilitated by the duodenoscopy. After retraction of the endoscope, a double lumen perfusion catheter (home made from PVC tubing, inner diameter 2.5 mm, outer diameter 4 mm) was inserted over the guide wire into the proximal small intestine with the tip of one lumen, which served as an injection port, located in the proximal descending duodenum and the tip of the second lumen, with three small holes in the last 3 cm to serve as a sample port, 40 cm distally from the injection port. After positioning, the guide wire was removed. The whole procedure was performed under short interval fluoroscopic control. To check positioning of the injection port in the duodenum throughout the experiment, intra-luminal pH was continuously measured at 5 cm proximal of the injection port by a pH electrode (Single Crystal Antimony Multi-Use pH Catheter, Synectics Medical, Queluz, Portugal). The 40-cm segment of the proximal small intestine was perfused with saline at 10 ml/min using a peristaltic pump for 195 min to obtain steady state conditions. After reaching steady state, 0.055M glutamine in saline (10 ml/min) was perfused for 4 hours. All ingoing solutions were kept on 37 degrees C in a water bath. Fifteen minutes after the perfusion experiment, the perfusion catheter was removed and a gastroduodenoscopy was performed to obtain tissue samples from the same intestinal region after the perfusion. Biopsies were imbedded in tissue-tek (Electron Microscopy Sciences, Washington, PA, USA), snap-frozen in melting isopentane and liquid nitrogen respectively, and stored at -80ºC until analysis.

The RNA extraction was performed using Sigma GenElute Mammalian Total RNA Miniprep Kit (RTN-70) (Sigma-Aldrich, Oakville, ON, Canada)

Labelled cRNA was prepared using the BioArray High Yield RNA Transcript Labelling Kit (Enzo, Farmingdale, NY, USA).

Fifteen µg of cRNA was fragmented at 94 degrees Celsius for 35 min in a fragmentation buffer containing 40 mM Tris-acetate pH 8.1, 100 mM KOAc, 30 mM MgOAc. Prior to hybridisation, the fragmented cRNA in a 6xSSPE-T hybridisation buffer (1 M NaCl, 10 mM Tris pH 7.6, 0.005% Triton), was heated to 94 degrees C for 5 min and subsequently to 45 degrees C for 5 min before loading onto the Affymetrix HG U133A probe array cartridge. The probe array was then incubated for 16 h at 45 degrees C at constant rotation (60 rpm). The washing and staining procedure was performed in the Affymetrix Fluidics Station. The probe array was exposed to 10 washes in 6xSSPE-T at 25 degrees C followed by 4 washes in 0.5xSSPE-T at 50 degrees C. The biotinylated cRNA was stained with a streptavidin-phycoerythrin conjugate, final concentration 2 mg/ml (Molecular Probes, Eugene, OR) in 6xSSPE-T for 30 min at 25 degrees C, followed by 10 washes in 6xSSPE-T at 25 degrees C. Subsequently, an antibody amplification step was performed, using normal goat IgG as blocking reagent, final concentration 0.1 mg/ml (Sigma, Copenhagen, DK) and biotinylated anti-streptavidin antibody (goat), to obtain a final concentration of 3 mg/ml (Vector Laboratories, Burlingame, CA, USA). This was followed by a staining step with a streptavidin-phycoerythrin conjugate, final concentration 2 mg/ml (Molecular Probes, Eugene, OR) in 6xSSPE-T for 30 min at 25 degrees C, and 10 washes in 6xSSPE-T at 25 degrees C.

The probe arrays were scanned at 560 nm using a confocal laser-scanning microscope (Hewlett Packard GeneArray Scanner G2500A).

The definition significant up or down regulation of individual genes was based on p-values of the comparisons of the normalized signal intensities using Bayesian statistics in R/ Bioconductor.

Transcription profiling by array of human duodenal mucosa after treatment with glutamine or glucose

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