FREE SHIPPING over $40
OVER 10000 FIVE-STAR REVIEWS
5% OFF ALL SUBSCRIPTIONS
100% HAPPINESS GUARANTEE
Analyze Diet
0
Journal of equine science2017; 28(4); 135-141; doi: 10.1294/jes.28.135

Comparison of commercial RNA extraction kits and qPCR master mixes for studying gene expression in small biopsy tissue samples from the equine gastric epithelium.

Abstract: Gastric tissue biopsy and gene expression analysis are important tools for disease diagnosis and study of the physiology of the equine stomach. However, RNA extraction from gastric biopsy samples is a complex procedure because the samples contain low quantities of RNA and are contaminated with mucous protein and bacterial flora. The objectives of these studies were to compare the performance of RNA extraction methods and to investigate the sensitivity of commercial qPCR master mixes for gene expression analysis of gastric biopsy samples. Three commercial RNA extraction methods (TRIzol, GENEzol and MiniPrep) and four qPCR master mixes with SYBR green (qPCRBIO, KAPA, QuantiNova, and PerfeCTa) were compared. RNA qualification and quantitation were compared. Real-time PCR was used to compare qPCR master mixes. The results revealed that TRIzol and GENEzol obtained significantly higher yield of RNA (P<0.01) but that TRIzol had the highest contamination of protein and DNA (P<0.05). Conversely, MiniPrep resulting in a significantly higher purification of RNA (P<0.05) but provided the lowest yield of RNA (P<0.01). For PCR master mixes, KAPA was significantly (P<0.05) more sensitive than other qPCR kits for all amounts of DNA template, particularly at the lowest amount of cDNA. In conclusion, GENEzol is the best method to obtain a high RNA yield and purification and it is more cost-effective than the others as well. Regarding the qPCR master mixes, KAPA SYBR qPCR Master Mix (2x) Universal is superior to the other tested master mixes for studying gene expression in equine gastric biopsies.
Get Full Text
Publication Date: 2017-12-16 PubMed ID: 29270070PubMed Central: PMC5735310DOI: 10.1294/jes.28.135Google Scholar: Lookup
The Equine Research Bank provides access to a large database of publicly available scientific literature. Inclusion in the Research Bank does not imply endorsement of study methods or findings by Mad Barn.
LinkedInCopy
  • Journal Article

Summary

This research summary has been generated with artificial intelligence and may contain errors and omissions. Refer to the original study to confirm details provided. Submit correction.

The research paper evaluates and compares the efficiency of various commercial RNA extraction methods and qPCR master mixes used in studying gene expression from small equine gastric biopsy samples. The results revealed that GENEzol produced high RNA yield and purification with more cost-effectiveness and that the KAPA SYBR qPCR Master Mix (2x) Universal outperformed other master mixes in analyzing gene expression.

RNA Extraction Methods Tested

  • The comparison involved three notable RNA extraction methods commonly used in gene expression analysis: TRIzol, GENEzol, and MiniPrep.
  • These extraction methods were evaluated based on yield and contamination levels of the resulting RNA.
  • The complexity of RNA extraction in this context is intensified due to the low RNA quantities in gastric biopsy samples, as well as the presence of mucous protein and bacterial flora that can cause contamination.

Results of RNA Extraction Comparison

  • The analysis revealed that TRIzol and GENEzol produced a significantly higher yield of RNA compared to MiniPrep.
  • However, TRIzol was found to have the highest rates of protein and DNA contamination.
  • Conversely, MiniPrep achieved a high level of RNA purification but provided the least RNA yield.
  • Considering the yield and level of purity, GENEzol was found to be the best RNA extraction method among the three, and was also more cost-effective.

Comparison of qPCR Master Mixes

  • The study also compared four different commercial qPCR master mixes (qPCRBIO, KAPA, QuantiNova, and PerfeCTa), which are essential in conducting a real-time Polymerase Chain Reaction (PCR) for gene expression analysis.
  • The sensitivity of these mixes was assessed using different DNA template amounts.

Results of qPCR Master Mixes Comparison

  • The results demonstrated that KAPA offered significantly higher sensitivity than the other tested mixes, especially with the lowest amount of cDNA.
  • This finding suggests that KAPA SYBR qPCR Master Mix (2x) Universal is the best choice for gene expression studies in equine gastric biopsies compared to the others.

Overall, this research provides critical insights for researchers working on gene expression analysis from gastric biopsy samples in horses. By identifying the most effective and cost-efficient RNA extraction method and qPCR master mix, this study contributes to improving the accuracy and efficiency of such advanced studies.

Cite This Article

APA
Tesena P, Korchunjit W, Taylor J, Wongtawan T. (2017). Comparison of commercial RNA extraction kits and qPCR master mixes for studying gene expression in small biopsy tissue samples from the equine gastric epithelium. J Equine Sci, 28(4), 135-141. https://doi.org/10.1294/jes.28.135

Publication

Journal of equine science
ISSN: 1340-3516
NlmUniqueID: 9503751
Country: Japan
Language: English
Volume: 28
Issue: 4
Pages: 135-141

Researcher Affiliations

Tesena, Parichart
  • Department of Clinical Science and Public Health, Faculty of Veterinary Science, Mahidol University, Nakhon Pathom 73170, Thailand.
Korchunjit, Wasamon
  • Laboratory of Cellular Biomedicine and Veterinary Medicine, Faculty of Veterinary Science, Mahidol University, Nakhon Pathom 73170, Thailand.
Taylor, Jane
  • Biomedical Teaching Organisation, Edinburgh Medical School, Biomedical Sciences, University of Edinburgh, Edinburgh EH8 9AG, U.K.
Wongtawan, Tuempong
  • Laboratory of Cellular Biomedicine and Veterinary Medicine, Faculty of Veterinary Science, Mahidol University, Nakhon Pathom 73170, Thailand.
  • Department of Preclinic and Applied Animal Science, Faculty of Veterinary Science, Mahidol University, Nakhon Pathom 73170, Thailand.

References

This article includes 20 references
  1. Arezi B, Xing W, Sorge JA, Hogrefe HH. Amplification efficiency of thermostable DNA polymerases.. Anal Biochem 2003 Oct 15;321(2):226-35.
    pubmed: 14511688doi: 10.1016/s0003-2697(03)00465-2google scholar: lookup
  2. Chomczynski P, Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction.. Anal Biochem 1987 Apr;162(1):156-9.
    pubmed: 2440339doi: 10.1006/abio.1987.9999google scholar: lookup
  3. Dong HJ, Ho H, Hwang H, Kim Y, Han J, Lee I, Cho S. Diversity of the Gastric Microbiota in Thoroughbred Racehorses Having Gastric Ulcer.. J Microbiol Biotechnol 2016 Apr 28;26(4):763-74.
    pubmed: 26809803doi: 10.4014/jmb.1507.07054google scholar: lookup
  4. Korchunjit V, Kaeoket K, Kitiyanant Y, Wongtawan T. Rapid Single Cell Typing using SYBR® Green Real-Time PCR Together with Melt Curve Analysis for Sex Identification of Porcine Sperm. Thai J. Vet. Med 44: 41–48.
  5. Marley LK, Repenning P, Frank CB, Hackett ES, Nout-Lomas YS. Transendoscopic Electrosurgery for Partial Removal of a Gastric Adenomatous Polyp in a Horse.. J Vet Intern Med 2016 Jul;30(4):1351-5.
    pmc: PMC5089670pubmed: 27238860doi: 10.1111/jvim.13979google scholar: lookup
  6. Miura M, Tanigawa C, Fujii Y, Kaneko S. Comparison of six commercially-available DNA polymerases for direct PCR.. Rev Inst Med Trop Sao Paulo 2013 Nov-Dec;55(6):401-6.
    pmc: PMC4105087pubmed: 24213192doi: 10.1590/s0036-46652013000600005google scholar: lookup
  7. Morrissey NK, Bellenger CR, Ryan MT, Baird AW. Cyclooxygenase-2 mRNA expression in equine nonglandular and glandular gastric mucosal biopsy specimens obtained before and after induction of gastric ulceration via intermittent feed deprivation.. Am J Vet Res 2010 Nov;71(11):1312-20.
    pubmed: 21034322doi: 10.2460/ajvr.71.11.1312google scholar: lookup
  8. Norhazlin J, Nor-Ashikin MN, Hoh BP, Sheikh Abdul Kadir SH, Norita S, Mohd-Fazirul M, Wan-Hafizah WJ, Razif D, Rajikin MH, Abdullah B. Effect of DNase treatment on RNA extraction from preimplantation murine embryos.. Genet Mol Res 2015 Aug 28;14(3):10172-84.
    pubmed: 26345954doi: 10.4238/2015.august.28.1google scholar: lookup
  9. Pfaffl M.W. Quantification strategies in real-time PCR. pp. 87–112.
  10. Pietra M, Morini M, Perfetti G, Spadari A, Vigo P, Peli A. Comparison of endoscopy, histology, and cytokine mRNA of the equine gastric mucosa.. Vet Res Commun 2010 Jun;34 Suppl 1:S121-4.
    pubmed: 20473566doi: 10.1007/s11259-010-9401-6google scholar: lookup
  11. Purzycka J.K, Olewiecki I, Soltyszewski I, Pepinski W, Janica J. Efficiency comparison of seven different Taq polymerases used in hemogenetics. Int. Congr. Ser. 1288: 719–721.
  12. Rio DC, Ares M Jr, Hannon GJ, Nilsen TW. Purification of RNA using TRIzol (TRI reagent).. Cold Spring Harb Protoc 2010 Jun;2010(6):pdb.prot5439.
    pubmed: 20516177doi: 10.1101/pdb.prot5439google scholar: lookup
  13. Rodrigues NL, Doré M, Doucet MY. Validation of a transendoscopic glandular and nonglandular gastric biopsy technique in horses.. Equine Vet J 2009 Sep;41(7):631-5.
    pubmed: 19927580doi: 10.2746/042516409x424144google scholar: lookup
  14. Sellin Jeffries MK, Kiss AJ, Smith AW, Oris JT. A comparison of commercially-available automated and manual extraction kits for the isolation of total RNA from small tissue samples.. BMC Biotechnol 2014 Nov 14;14:94.
    pmc: PMC4239376pubmed: 25394494doi: 10.1186/s12896-014-0094-8google scholar: lookup
  15. Sultan M, Amstislavskiy V, Risch T, Schuette M, Dökel S, Ralser M, Balzereit D, Lehrach H, Yaspo ML. Influence of RNA extraction methods and library selection schemes on RNA-seq data.. BMC Genomics 2014 Aug 11;15(1):675.
    pmc: PMC4148917pubmed: 25113896doi: 10.1186/1471-2164-15-675google scholar: lookup
  16. Tesena P, Yingchutraku Y, Wongtawan T, Angkanaporn K. Electrophoresis separation of serum protein in Thoroughbred horse with gastric ulcer syndrome. pp. 316−318.
  17. Tolosa JM, Schjenken JE, Civiti TD, Clifton VL, Smith R. Column-based method to simultaneously extract DNA, RNA, and proteins from the same sample.. Biotechniques 2007 Dec;43(6):799-804.
    pubmed: 18251257doi: 10.2144/000112594google scholar: lookup
  18. Watchrarat K, Korchunjit W, Buranasinsup S, Taylor J, Ritruechai P, Wongtawan T. MEM α Promotes Cell Proliferation and Expression of Bone Marrow Derived Equine Mesenchymal Stem Cell Gene Markers but Depresses Differentiation Gene Markers. J. Equine Vet. Sci. 50: 8–14.
  19. Wex T, Treiber G, Lendeckel U, Malfertheiner P. A two-step method for the extraction of high-quality RNA from endoscopic biopsies.. Clin Chem Lab Med 2003 Aug;41(8):1033-7.
    pubmed: 12964810doi: 10.1515/cclm.2003.159google scholar: lookup
  20. Ye J, Coulouris G, Zaretskaya I, Cutcutache I, Rozen S, Madden TL. Primer-BLAST: a tool to design target-specific primers for polymerase chain reaction.. BMC Bioinformatics 2012 Jun 18;13:134.
    pmc: PMC3412702pubmed: 22708584doi: 10.1186/1471-2105-13-134google scholar: lookup

Citations

This article has been cited 6 times.
  1. Supokawej A, Korchunjit W, Wongtawan T. The combination of BMP12 and KY02111 enhances tendon differentiation in bone marrow-derived equine mesenchymal stromal cells (BM-eMSCs). J Equine Sci 2022 Jul;33(2):19-26.
    doi: 10.1294/jes.33.19pubmed: 35847484google scholar: lookup
  2. Amirouche A, Ait-Ali D, Nouri H, Boudrahme-Hannou L, Tliba S, Ghidouche A, Bitam I. TRIzol-based RNA extraction for detection protocol for SARS-CoV-2 of coronavirus disease 2019. New Microbes New Infect 2021 May;41:100874.
    doi: 10.1016/j.nmni.2021.100874pubmed: 33815807google scholar: lookup
  3. Busi SB, Pramateftaki P, Brandani J, Fodelianakis S, Peter H, Halder R, Wilmes P, Battin TJ. Optimised biomolecular extraction for metagenomic analysis of microbial biofilms from high-mountain streams. PeerJ 2020;8:e9973.
    doi: 10.7717/peerj.9973pubmed: 33194372google scholar: lookup
  4. Déri MT, Kiss ÁF, Tóth K, Paulik J, Sárváry E, Kóbori L, Monostory K. End-stage renal disease reduces the expression of drug-metabolizing cytochrome P450s. Pharmacol Rep 2020 Dec;72(6):1695-1705.
    doi: 10.1007/s43440-020-00127-wpubmed: 32638224google scholar: lookup
  5. Juang DS, Berry SM, Li C, Lang JM, Beebe DJ. Centrifugation-Assisted Immiscible Fluid Filtration for Dual-Bioanalyte Extraction. Anal Chem 2019 Sep 17;91(18):11848-11855.
    doi: 10.1021/acs.analchem.9b02572pubmed: 31411020google scholar: lookup
  6. Chakravarthi VP, Christenson LK. Wet Lab Techniques for the Functional Analysis of Circular RNA. Cells 2025 Dec 3;14(23).
    doi: 10.3390/cells14231920pubmed: 41369410google scholar: lookup
Subscribe to our newsletter
Join 100,151 horse owners like you who receive our email updates on horse health and nutrition.