Selection of Optimal Extraction and RT-PCR Protocols for Stool RNA Detection of Colorectal Cancer Associated Immune Genes

Overview

Journal Sci Rep

Specialty Science

Date 2024 Nov 10

PMID 39523395

Authors

Thura Akrem Omran

Inger Line Madso

Per Christian Saether

Vahid Bemanian

Hege Smith Tunsjo

Affiliations

Soon will be listed here.

Abstract

There is a growing interest in using fecal mRNA transcripts as biomarkers for non-invasive detection of colorectal cancer (CRC). The following study compares different RNA extraction and reverse transcription PCR (RT-PCR) methods for mRNA detection in stool and identifies a robust and sensitive protocol. A combination of the Stool total RNA purification kit (Norgen) and the Superscript III one-step RT-PCR kit (Invitrogen) provided high RNA purity and sensitive and consistent mRNA detection, making them well-suited candidates for large-scale studies. We tested the protocol by detecting the mRNA of several immune genes (CXCL1, IL8, IL1B, IL6, PTGS2, and SPP1) in 22 CRCs, 24 adenomatous polyps, and 22 control stool samples. All these inflammatory markers, except for CXCL1, showed a strong association with CRC. Cancer stool samples showed increased levels of IL1B, IL8, and PTGS2 transcripts compared to polyp and control groups. Thus, this work supports the potential use of fecal mRNA as biomarkers for CRC detection.

References

Bustin S, Benes V, Garson J, Hellemans J, Huggett J, Kubista M . The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments. Clin Chem. 2009; 55(4):611-22. DOI: 10.1373/clinchem.2008.112797. View

Borowczak J, Szczerbowski K, Maniewski M, Kowalewski A, Janiczek-Polewska M, Szylberg A . The Role of Inflammatory Cytokines in the Pathogenesis of Colorectal Carcinoma-Recent Findings and Review. Biomedicines. 2022; 10(7). PMC: 9312930. DOI: 10.3390/biomedicines10071670. View

Zhang X, Yang X, Zhang Y, Liu X, Zheng G, Yang Y . Direct serum assay for cell-free bmi-1 mRNA and its potential diagnostic and prognostic value for colorectal cancer. Clin Cancer Res. 2014; 21(5):1225-33. DOI: 10.1158/1078-0432.CCR-14-1761. View

Virtanen P, Gommers R, Oliphant T, Haberland M, Reddy T, Cournapeau D . SciPy 1.0: fundamental algorithms for scientific computing in Python. Nat Methods. 2020; 17(3):261-272. PMC: 7056644. DOI: 10.1038/s41592-019-0686-2. View

Mutter G, Zahrieh D, Liu C, Neuberg D, Finkelstein D, Baker H . Comparison of frozen and RNALater solid tissue storage methods for use in RNA expression microarrays. BMC Genomics. 2004; 5:88. PMC: 534099. DOI: 10.1186/1471-2164-5-88. View

Gordon S, Pluddemann A . Macrophage Clearance of Apoptotic Cells: A Critical Assessment. Front Immunol. 2018; 9:127. PMC: 5797608. DOI: 10.3389/fimmu.2018.00127. View

Patterson A, Watson A . Deciphering the Complex Signaling Systems That Regulate Intestinal Epithelial Cell Death Processes and Shedding. Front Immunol. 2017; 8:841. PMC: 5513916. DOI: 10.3389/fimmu.2017.00841. View

Livak K, Schmittgen T . Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods. 2002; 25(4):402-8. DOI: 10.1006/meth.2001.1262. View

Hamaya Y, Yoshida K, Takai T, Ikuma M, Hishida A, Kanaoka S . Factors that contribute to faecal cyclooxygenase-2 mRNA expression in subjects with colorectal cancer. Br J Cancer. 2010; 102(5):916-21. PMC: 2833255. DOI: 10.1038/sj.bjc.6605564. View

10.

Daulagala A, Bridges M, Kourtidis A . E-cadherin Beyond Structure: A Signaling Hub in Colon Homeostasis and Disease. Int J Mol Sci. 2019; 20(11). PMC: 6600153. DOI: 10.3390/ijms20112756. View

11.

Beaulieu J, Herring E . Toward a Stool mRNA-Based Assay for Detection of Colorectal Cancer Lesions. Gastroenterology. 2019; 158(3):793. DOI: 10.1053/j.gastro.2019.10.033. View

12.

Herring E, Kanaoka S, Tremblay E, Beaulieu J . Droplet digital PCR for quantification of in a stool mRNA assay for the detection of colorectal cancers. World J Gastroenterol. 2017; 23(16):2891-2898. PMC: 5413784. DOI: 10.3748/wjg.v23.i16.2891. View

13.

Wang X, Yang F, Xu G, Zhong S . The roles of IL-6, IL-8 and IL-10 gene polymorphisms in gastric cancer: A meta-analysis. Cytokine. 2018; 111:230-236. DOI: 10.1016/j.cyto.2018.08.024. View

14.

Qi J, Sun H, Zhang Y, Wang Z, Xun Z, Li Z . Single-cell and spatial analysis reveal interaction of FAP fibroblasts and SPP1 macrophages in colorectal cancer. Nat Commun. 2022; 13(1):1742. PMC: 8976074. DOI: 10.1038/s41467-022-29366-6. View

15.

Krzystek-Korpacka M, Diakowska D, Kapturkiewicz B, Bebenek M, Gamian A . Profiles of circulating inflammatory cytokines in colorectal cancer (CRC), high cancer risk conditions, and health are distinct. Possible implications for CRC screening and surveillance. Cancer Lett. 2013; 337(1):107-14. DOI: 10.1016/j.canlet.2013.05.033. View

16.

Beaulieu J, Herring E, Kanaoka S, Tremblay E . Use of integrin alpha 6 transcripts in a stool mRNA assay for the detection of colorectal cancers at curable stages. Oncotarget. 2016; 7(12):14684-92. PMC: 4924744. DOI: 10.18632/oncotarget.7407. View

17.

Oladipo O, Conlon S, OGrady A, Purcell C, Wilson C, Maxwell P . The expression and prognostic impact of CXC-chemokines in stage II and III colorectal cancer epithelial and stromal tissue. Br J Cancer. 2011; 104(3):480-7. PMC: 3049559. DOI: 10.1038/sj.bjc.6606055. View

18.

Bray F, Laversanne M, Sung H, Ferlay J, Siegel R, Soerjomataram I . Global cancer statistics 2022: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin. 2024; 74(3):229-263. DOI: 10.3322/caac.21834. View

19.

Grotzer M, Patti R, Geoerger B, Eggert A, CHOU T, Phillips P . Biological stability of RNA isolated from RNAlater-treated brain tumor and neuroblastoma xenografts. Med Pediatr Oncol. 2000; 34(6):438-42. DOI: 10.1002/(sici)1096-911x(200006)34:6<438::aid-mpo12>3.0.co;2-q. View

20.

Lin J, Perdue L, Henrikson N, Bean S, Blasi P . Screening for Colorectal Cancer: Updated Evidence Report and Systematic Review for the US Preventive Services Task Force. JAMA. 2021; 325(19):1978-1998. DOI: 10.1001/jama.2021.4417. View