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The intestinal fragments that were freshly collected and immediately frozen in liquid nitrogen were stored at -170°C until processing to study the expression of the c-fos and c-jun genes in association with cell proliferation and apoptosis. First, total RNA was extracted from intestinal samples using TRIZOL™ reagent (Invitrogen, Carlsbad, CA, USA). Approximately 100 mg of intestine was fragmented in a tissue mill (Mikro Dismembrator U, Sartorius AG, Goettingen, Germany) after the addition of liquid nitrogen and was then homogenized in 700 μl of TRIZOL solution according to the manufacturer’s protocol.
The total RNA concentration was measured using a spectrophotometer (BioPhotometer, Eppendorf AG, Germany) at 260 nm, and RNA quality was assessed by calculating the 260 nm/280 nm absorbance ratio. One microgram of RNA was subjected to agarose gel electrophoresis to assess its integrity by visualizing the fragments corresponding to 18S and 28S ribosomal RNAs.
Complementary DNA (cDNA) was prepared from 2 μg of total RNA via reverse transcription using 200 U of SuperScript III RNase H-RT (Invitrogen) and oligo(dT)s as primers. The resulting cDNA solution was stored at -20°C.
Gene expression was evaluated by two methods, conventional semiquantitative RT-polymerase chain reaction (PCR) and real-time RT-PCR analyses, and the results of these two methods were compared.