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.
Publication Date: 2017-12-16 PubMed ID: 29270070PubMed Central: PMC5735310DOI: 10.1294/jes.28.135Google Scholar: Lookup
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Summary
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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
Researcher Affiliations
- Department of Clinical Science and Public Health, Faculty of Veterinary Science, Mahidol University, Nakhon Pathom 73170, Thailand.
- Laboratory of Cellular Biomedicine and Veterinary Medicine, Faculty of Veterinary Science, Mahidol University, Nakhon Pathom 73170, Thailand.
- Biomedical Teaching Organisation, Edinburgh Medical School, Biomedical Sciences, University of Edinburgh, Edinburgh EH8 9AG, U.K.
- 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.
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Citations
This article has been cited 6 times.- 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.
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