Analysis of intestinal adaptation gene expression by cDNA expression arrays

JPEN: Journal of Parenteral and Enteral Nutrition,  Nov/Dec 2000  by Erwin, Christopher R,  Falcone, Richard A Jr,  Stern, Lawrence E,  Kemp, Christopher J,  Warner, Brad W

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cDNA Synthesis and Hybridization

The [alpha^sup 32^P]dATP-labeled cDNA probes for sham and SBR samples were made from poly(A+) RNA by reverse transcriptase. The cDNA probes for sham and SBR were hybridized to separate array membranes at 68 deg C. The probe synthesis and hybridization were performed using reagents supplied in the Clontech Atlas mouse cDNA expression array kit (Clontech Laboratories, Palo Alto, CA) following the manufacturer's instructions. After hybridization, the arrays were exposed to a phosphorimaging screen at room temperature for various times, and differences between the two blots were quantified using a Molecular Dynamics PhosphorImager system and ImageQuaNT software (Molecular Dynamics, Sunnyvale, CA). The arrays then were exposed to autoradiography film at -70 deg C with an intensifying screen. Finally, the array membranes were boiled for 5 minutes in 0.5% sodium dodecyl sulfate (SDS) to strip the cDNA probe and allow for reuse of the arrays.

Semiquantitative RT-PCR

Total RNA from sham and SBR (after DNase I treatment) was used to make cDNA by reverse transcriptale (RT) reactions.8 Using the known DNA sequence for the positive clones, polymerase chain reaction (PCR) primer pairs were designed (Table I). For some primer pairs, proprietary sequence information was purchased from Clontech Laboratories, PCR products were analyzed on agarose gels and band density determined using a Kodak digital camera and image analysis software (Eastman Kodak, Rochester, NY).

Northern Blot Analysis

For sham and SBR, 1 (mu)g of poly(A+) RNA was run on a denaturing agarose/formaldehyde gel, blotted onto a nylon membrane, baked under vacuum, and stored at -20 deg C for subsequent use. PCR product bands of the correct size were isolated, labeled with ^sup 32^p, hybridized to the blot, washed, and exposed to a phosphorimaging screen or autoradiography film. The Northern blots were quantified using a Molecular Dynamics PhosphorImager system and ImageQuaNT software. Blots were stripped and reprobed for beta-actin DNA as a control.

RESULTS

Intestinal Adaptation

Previous studies using this marine model of SBR have shown characteristic morphologic and biochemical changes consistent with intestinal adaptation7,9 that are maximal 3 days after SBR.10,11 In this study, significant increase in ileal wet weight and RNA concentration was observed after SBR (Figs. 1A,B).

Expression Profile for Adaptation

cDNA probes synthesized from RNA isolated from the ilea of Sham and SBR mice were used to screen mouse cDNA expression arrays (Fig. 2). The array results are summarized in Table II both by overall differences between sham and SBR, and by functional class.

RT-PCR Confirmation

RT-PCR was used to confirm differences in expression between sham and SBR for three genes with beta-actin served as an internal control (Fig. 3). The RT-PCR results confirmed the expression changes detected by the array, although the magnitude of the change tended to be greater by RT-PCR when compared with the array.