J Anim Sci. 2023 Jun 18:skad202. doi: 10.1093/jas/skad202. Online ahead of print.
ABSTRACT
Dextran sodium sulfate (DSS) is commonly used to induce intestinal (i.e., colonic) inflammation in a variety of animal models. However, DSS is known to cause interference when using quantitative-real time polymerase chain reaction (qRT-PCR) methods, thereby invalidating accurate and precise measurement of tissue gene expression. Therefore, the goal of this study was to determine whether different mRNA purification methods would reduce DSS-interference. Colonic tissue samples were collected at postnatal d (PND) 27 or 28 from pigs that had not been administered DSS (Control), and 2 independent groups of pigs that received 1.25 g of DSS/kg of BW/d (DSS-1 and DSS-2) from PND 14-18. Tissue samples collected were subsequently stratified into 3 purification methods (i.e., 9 total treatment x method combinations), including: 1) No Purification, 2) purification with lithium chloride (LiCl), or 3) purification using Spin Column filtration. All data were analyzed using a one-way ANOVA in the Mixed procedure of SAS. The average ribonucleic acid (RNA) concentrations across all treatments were between 1,300 and 1,800 µg/µL for all 3 in vivo groups. Although there were statistical differences among purification methods, the 260/280 ratio and 260/230 ratio fell between acceptable limits of 2.0-2.1 and 2.0-2.2, respectively, for all treatment groups. This confirms the RNA quality was adequate and not influenced by purification method in addition to suggesting the absence of phenol, salts, and carbohydrate contamination. For pigs in the Control group that did not receive DSS, qRT-PCR Ct values of 4 cytokines were achieved, though these values were not altered by purification method. For pigs that had undergone DSS dosing, those tissues subjected to either no purification or purification using LiCl did not generate applicable Ct values. However, when tissues derive from DSS-treated pigs underwent spin column purification, half of the samples from DSS-1 and DSS-2 groups generated appropriate Ct estimates. Therefore, spin column purification appeared to be more effective than LiCl purification, but no method was 100% effective, so caution should be exercised when interpreting gene expression results from studies where animals are exposed to DSS-induced colitis.
PMID:37330677 | DOI:10.1093/jas/skad202
