Dextran sodium sulfate inhibits the activities of both polymerase and reverse transcriptase: lithium chloride purification, a rapid and efficient technique to purify RNA

BMC Research Notes

Didier Merlin¹  Mark T Baker²  Hamed Laroui²  Fengyuan Chen³  Emilie Viennois² 

[1] Veterans Affairs Medical Center, Decatur GA 30033, USA;Department of Biology, Center for Diagnostics and Therapeutics, Georgia State University, Atlanta GA 30303, USA;Department of Gastroenterology, Shanghai Fifth People’s Hospital, Fudan University, 128 Ruili Road, 200240, Shanghai, People’s Republic of China

关键词: RNA;    Lithium chloride;    Reverse transcriptase;    DNA-polymerase;    DSS;   

Others  :  1141690
DOI  :  10.1186/1756-0500-6-360

 received in 2013-01-31, accepted in 2013-09-03,  发布年份 2013

【 摘 要 】

Background

Dextran sodium sulfate (DSS) is commonly used in mouse studies to induce a very reproducible colitis that effectively mimics the clinical and histological features of human inflammatory bowel disease (IBD) patients, especially ulcerative colitis. However, the mechanisms of action of DSS remain poorly understood, and observations by our laboratory and other groups indicate that DSS contamination of colonic tissues from DSS-treated mice potently inhibits the quantitative reverse-transcription polymerase chain reaction (qRT-PCR) amplification of mRNA.

Results

A prior study used poly-A-mediated mRNA purification to remove DSS from RNA extracts, but we herein report a second efficient and cost-effective approach to counteract this inhibition, using lithium chloride precipitation to entirely remove DSS from RNAs. We also explored how DSS interferes with qRT-PCR process, and we report for the first time that DSS can alter the binding of reverse transcriptase to previously primed RNA and specifically inhibits the enzymatic activities of reverse transcriptase and Taq polymerase in vitro. This likely explains why DSS-treated colonic RNA is not suitable to qRT-PCR amplification without a previous purification step.

Conclusion

In summary, we provide a simple method to remove DSS from colonic RNAs, and we demonstrate for the first time that DSS can inhibit the activities of both polymerase and reverse transcriptase. In order to reliably analyze gene expression in the colonic mucosa of DSS-treated mice, the efficiency rate of qRT-PCR must be the same between all the different experimental groups, including the water-treated control group, suggesting that whatever the duration and the percentage of the DSS treatment, RNAs must be purified.

【 授权许可】

   
2013 Viennois et al.; licensee BioMed Central Ltd.

【 图 表 】

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【 参考文献 】

• [1]Dieleman LA, Palmen MJ, Akol H, Bloemena E, Pena AS, Meuwissen SG, Van Rees EP: Chronic experimental colitis induced by dextran sulphate sodium (DSS) is characterized by Th1 and Th2 cytokines. Clin Exp Immunol 1998, 114(3):385-391.
• [2]Elson CO, Sartor RB, Tennyson GS, Riddell RH: Experimental models of inflammatory bowel disease. Gastroenterology 1995, 109(4):1344-1367.
• [3]Ohkusa T: Production of experimental ulcerative colitis in hamsters by dextran sulfate sodium and changes in intestinal microflora. Nihon Shokakibyo Gakkai Zasshi 1985, 82(5):1327-1336.
• [4]Okayasu I, Hatakeyama S, Yamada M, Ohkusa T, Inagaki Y, Nakaya R: A novel method in the induction of reliable experimental acute and chronic ulcerative colitis in mice. Gastroenterology 1990, 98(3):694-702.
• [5]Cooper HS, Murthy SNS, Shah RS, Sedergran DJ: Clinicopathological study of dextran sulfate sodium experimental murine colitis. Lab Invest 1993, 69(2):238-249.
• [6]Melgar S, Karlsson L, Rehnstrom E, Karlsson A, Utkovic H, Jansson L, Michaelsson E: Validation of murine dextran sulfate sodium-induced colitis using four therapeutic agents for human inflammatory bowel disease. Int Immunopharmacol 2008, 8(6):836-844.
• [7]De Robertis M, Massi E, Poeta ML, Carotti S, Morini S, Cecchetelli L, Signori E, Fazio VM: The AOM/DSS murine model for the study of colon carcinogenesis: from pathways to diagnosis and therapy studies. J Carcinog 2011, 10:9.
• [8]Kanneganti M, Mino-Kenudson M, Mizoguchi E: Animal models of colitis-associated carcinogenesis. J Biomed Biotechnol 2011, 2011:342637.
• [9]Perse M, Cerar A: Dextran sodium sulphate colitis mouse model: traps and tricks. J Biomed Biotechnol 2012, 2012:718617.
• [10]Yan Y, Kolachala V, Dalmasso G, Nguyen H, Laroui H, Sitaraman SV, Merlin D: Temporal and spatial analysis of clinical and molecular parameters in dextran sodium sulfate induced colitis. PLoS One 2009, 4(6):e6073.
• [11]Laroui H, Ingersoll SA, Liu HC, Baker MT, Ayyadurai S, Charania MA, Laroui F, Yan Y, Sitaraman SV, Merlin D: Dextran sodium sulfate (DSS) induces colitis in mice by forming nano-lipocomplexes with medium-chain-length fatty acids in the colon. PLoS One 2012, 7(3):e32084.
• [12]Wirtz S, Neufert C, Weigmann B, Neurath MF: Chemically induced mouse models of intestinal inflammation. Nat Protoc 2007, 2(3):541-546.
• [13]Wirtz S, Neurath MF: Mouse models of inflammatory bowel disease. Adv Drug Deliv Rev 2007, 59(11):1073-1083.
• [14]Kerr TA, Ciorba MA, Matsumoto H, Davis VR, Luo J, Kennedy S, Xie Y, Shaker A, Dieckgraefe BK, Davidson NO: Dextran sodium sulfate inhibition of real-time polymerase chain reaction amplification: a poly-A purification solution. Inflamm Bowel Dis 2012, 18(2):344-348.
• [15]Charania MA, Ayyadurai S, Ingersoll SA, Xiao B, Viennois E, Yan Y, Laroui H, Sitaraman SV, Merlin D: Intestinal epithelial CD98 synthesis specifically modulates expression of colonic microRNAs during colitis. Am J Physiol Gastrointest Liver Physiol 2012, 302(11):G1282-1291.
• [16]Chassaing B, Srinivasan G, Delgado MA, Young AN, Gewirtz AT, Vijay-Kumar M: Fecal lipocalin 2, a sensitive and broadly dynamic non-invasive biomarker for intestinal inflammation. PLoS One 2012, 7(9):e44328.
• [17]Dalmasso G, Nguyen HT, Ingersoll SA, Ayyadurai S, Laroui H, Charania MA, Yan Y, Sitaraman SV, Merlin D: The PepT1-NOD2 signaling pathway aggravates induced colitis in mice. Gastroenterology 2011, 141(4):1334-1345.
• [18]Nguyen HT, Dalmasso G, Torkvist L, Halfvarson J, Yan Y, Laroui H, Shmerling D, Tallone T, D'Amato M, Sitaraman SV, et al.: CD98 expression modulates intestinal homeostasis, inflammation, and colitis-associated cancer in mice. J Clin Invest 2011, 121(5):1733-1747.
• [19]Miyazawa F, Olijnyk OR, Tilley CJ, Tamaoki T: Interactions between dextran sulfate and Escherichia coli ribosomes. Biochim Biophys Acta 1967, 145(1):96-104.
• [20]Fellig J, Wiley CE: The inhibition of pancreatic ribonuclease by anionic polymers. Arch Biochem Biophys 1959, 85:313-316.
• [21]Philipson L, Zetterqvist O: The presence of DNA in human erythrocyte membranes. Biochim Biophys Acta 1964, 91:171-173.
• [22]Charania MA, Laroui H, Liu H, Viennois E, Ayyadurai S, Xiao B, Ingersoll SA, Kalman D, Merlin D: Intestinal epithelial CD98 directly modulates the innate host response to enteric bacterial pathogens. Infect Immun 2013, 81(3):923-934.
• [23]Laroui H, Yan Y, Narui Y, Ingersoll SA, Ayyadurai S, Charania MA, Zhou F, Wang B, Salaita K, Sitaraman SV, et al.: L-Ala-gamma-D-Glu-meso-diaminopimelic acid (DAP) interacts directly with leucine-rich region domain of nucleotide-binding oligomerization domain 1, increasing phosphorylation activity of receptor-interacting serine/threonine-protein kinase 2 and its interaction with nucleotide-binding oligomerization domain 1. J Biol Chem 2011, 286(35):31003-31013.
• [24]Yao X, Li X, Toledo F, Zurita-Lopez C, Gutova M, Momand J, Zhou F: Sub-attomole oligonucleotide and p53 cDNA determinations via a high-resolution surface plasmon resonance combined with oligonucleotide-capped gold nanoparticle signal amplification. Anal Biochem 2006, 354(2):220-228.