ARTICLE IN PRESS 4
J. Yu et al. / Comparative Biochemistry and Physiology, Part A xxx (2010) xxx–xxx
Fig. 4. Photomicrographs of hematoxylin- and eosin-stained sections of the pig small intestine from heat treated and control animals after 3 days of treatment (200× magniﬁcation). A and B) Control and heat treated duodenums, respectively; C and D) control and heat treated jejunums respectively; E and F) control and heat treated ileums, respectively. Severe damage to the intestinal villi is apparent, with desquamation at the tips of the intestinal villi and exposure of the lamina propria. Abnormal microstructures are indicated with arrowheads. Scale bar 100 μm.
2.6. Validation of HSP90, HSP70, HSP27, EGF and EGFR mRNA in the pig jejunum by real-time PCR Expression of HSP70, HSP90, HSP27, EGF and EGFR were quantitatively determined using real-time PCR. Quantitative PCR analysis was carried out using the DNA Engine Mx3000P® ﬂuorescence detection system against a double-stranded DNA-speciﬁc ﬂuorescent dye (Stratagene, USA) according to optimized PCR protocols. β-actin was ampliﬁed in parallel with the target genes providing a control. The cDNA was subjected to real-time RT-PCR using the primer pairs listed in Table 1. The PCR reaction system (20 μL) contained 10 μL of SYBR Green qPCR mix, 0.3 μL of reference dye, 1 μL of each primer (both 10 μmol/L), and 1 μL of cDNA template. Cycling conditions were 94 °C for 5 min, followed by 40 cycles of 94 °C for 30 s, 56 °C for 30 s and 72 °C for 40 s. Dissociation began with a step of 95 °C for 1 min,
and then the melting curve from 55 to 95 °C at 0.2 °C/s was monitored continuously by measuring ﬂuorescence. Expression levels were determined using the threshold cycle (CT) method as described by the manufacturer of the detection system. This method was applied to each gene by calculating the expression 2− ΔΔCT, where ΔΔCT is the sum of: [CTgene − CTβ-actin](Heat-stressed) − [CTgene − CTβ-actin](Control).
2.7. Statistical analysis All results are presented as the mean ± SD. Statistical analysis was performed by independent-sample T-tests using SPSS 12.0. A P-value of < 0.05 was considered signiﬁcant. Microarray analysis was conducted using a Molecule Annotation System (http://bioinfo. capitalbio.com/mas/).
Please cite this article as: Yu, J., et al., Effect of heat stress on the porcine small intestine: A morphological and gene expression study, Comp. Biochem. Physiol. A (2010), doi:10.1016/j.cbpa.2010.01.008
Published on Apr 5, 2010
Keywords: Heat stress Morphology Gene expression Electron microscope Microarray Small intestine Pig Article history: Received 26 November 20...