Abstract
We have developed a protocol to purify RNA from DSS (Dextran Sulfate Sodium)-treated mouse tissues. This method, which prevents downstream inhibition of q-RT-PCR observed in DSS-treated tissues, relies on successive precipitations with lithium chloride.
Keywords: Dextran sodium sulfate, Colitis, Lithium chloride, RNA, q-RT-PCR
Background
Dextran Sulfate Sodium (DSS) is very commonly used in laboratories to induce colitis in rodents. Specifically, it mimics the clinical and histological features of human Inflammatory Bowel Disease (IBD) with Ulcerative Colitis (UC) characteristics. DSS is diluted in the drinking water and penetrates tissues. We have observed that contamination of RNA extracts with DSS prevented successful subsequent amplification processes from the colon and small intestine, but also blood and other tissues obtained from DSS-treated animals. We had previously shown that the presence of DSS in the samples inhibited reverse transcription and polymerase chain reaction amplification (Viennois et al., 2013). This inhibitory effect was observed in a dose depended manner by Kerr et al. and they suggested a poly-A-purification based technique to remove DSS from total RNA extract (Kerr et al., 2012). We hereby propose another efficient and economical method for purifying total RNA extracts from DSS traces based on lithium chloride (LiCl) precipitations. This method has been extensively used in our laboratory as well as in others (Chassaing et al., 2012, Li et al., 2016); however, no attempt has been taken to document the procedure in detail. Therefore, we provide a detailed description of LiCl purification procedure of total RNA primarily isolated from DSS-treated murine tissue with another method (Trizol, Spin column-based nucleic acid purification…).
Materials and Reagents
Equipment
Procedure
Data analysis
Agarose gel electrophoresis demonstrates that, before LiCl precipitation, housekeeping gene 36B4 cDNA amplification products are obtained from non-DSS-treated mice as indicated by a clear band while amplification is inhibited in DSS-treated mice as indicated by the smear (Figure 1). After LiCl precipitation, cDNA obtained from non-DSS and DSS treated samples were both successfully amplified, as attested by a distinct band (Figure 1). Figure 1. LiCl purification allows amplification of cDNA from DSS-treated tissues. Total RNAs were extracted from colonic tissues obtained from mice treated with DSS diluted at 3% in the drinking water or water controls. After cDNA synthesis, qPCR for the housekeeping gene 36B4 was performed and the product of amplification was visualized by electrophoresis. The presence of a smear indicates that the amplification of 36B4 cDNA was inhibited in DSS-treated mice before LiCl purification. After purification of the DSS- and non-DSS RNA samples with LiCl, clear bands indicate that 36B4 cDNA was successfully amplified from both DSS- and non-DSS samples.
Notes
Recipes
Acknowledgments
EV is a recipient of the Career Development Award from the Crohn’s and Colitis Foundation. DM is a recipient of a Research Scientist Award from the Department of Veteran Affairs. This work was supported by grants from the National Institutes of Health of Diabetes and Digestive and Kidney (DK116306, DK107739; DK071594 to DM). We thank Samantha Spencer for proofreading the manuscript. This protocol was adapted from published work by Cathala et al. (1983) and Viennois et al. (2013). The authors declare no conflicts of interest within this work.
References
If you have any questions/comments about this protocol, you are highly recommended to post here. We will invite the authors of this protocol as well as some of its users to address your questions/comments. To make it easier for them to help you, you are encouraged to post your data including images for the troubleshooting.