PMC

Optimization of RT-PCR reactions for Gklf and β-actin. cDNA derived from 125 ng total RNA from wild-type mouse intestine was amplified for 21–39 cycles using primers for Gklf (A) and 17–35 cycles using primers for β-actin (B). The intensity of the RT-PCR bands, as measured by quantitative densitometric tracings, as a function of the number of cycles is plotted. The arrows indicate the numbers of cycles selected for subsequent experiments for Gklf and β-actin, respectively.

Semi-quantitative RT-PCR of Gklf, β-actin and CK19 transcripts in Min and wild-type mouse intestines. RNA was extracted from the intestines of wild-type mice (WT) and the normal-appearing mucosa of Min mice (0), as well as the pooled adenomas of Min mouse intestines based on size: <3 mm, 3–6 mm, and >6 mm. cDNA produced from 125 ng total RNA was subject to PCR using primers for Gklf, β-actin and CK19. Products were resolved on a 1.5% agarose gel.

Semi-quantitative RT-PCR of GKLF and β-actin transcripts in control individuals and FAP patients. RNA was extracted from biopsied specimens of normal colon from two control individuals (left panel) and normal-appearing colonic mucosa (N) and adenomatous polyps (P) from three FAP patients (right panel). cDNA template made from 125 ng total RNA was subject to PCR using primers for human GKLF. To ensure even loading, the same amount of template was subjected to PCR using primers for β-actin.

MSP of genomic DNA from mouse tissues. Genomic DNA was extracted from normal-appearing intestinal mucosa and adenomas of different size of Min mice, and from the small intestine (SI) and liver of wild-type (WT) control littermates. Following treatment of the DNA with bisulfite, MSP was performed according to the protocol described in Section 2 using primers designed to amplify either methylated (M) or unmethylated (U) DNA present in the 5′-UTR of the mouse Gklf gene []. The expected product for the unmethylated DNA is 172 bp. As a control for methylated product, in vitro methylated mouse genomic DNA (IVD) was similarly analyzed by MSP and the methylated DNA product is 162 bp. Water was used as a negative control (No DNA).