In Silico and Validation Approaches for Optimum Conditions of Rattus norvegicus Target Gene qPCR Primers

https://doi.org/10.22146/jtbb.71765

Gracia Alice Victoria Pollo(1), Nyoman Yudi Antara(2), Firman Alamsyah(3), Rarastoeti Pratiwi(4*)

(1) Faculty of Biology, Universitas Gadjah Mada, Yogyakarta, 55281, Indonesia
(2) Faculty of Health, Universitas Kader Bangsa, Palembang, 30129, Indonesia
(3) Center for Medical Physics and Cancer Research, CTECH Laboratories EDWAR TECHNOLOGY, Tangerang, 15320, Indonesia
(4) Faculty of Biology, Universitas Gadjah Mada, Yogyakarta, 55281, Indonesia
(*) Corresponding Author

Abstract


The qPCR method requires an oligonucleotide pair to prime the amplification process. With the variety of qPCR reagent and primer options available, in silico and laboratory experimental validation approach was needed to validate the most suitable primer for prior use. This article aims to provide in silico analysis of actin alpha-2 smooth muscle (Acta2), fibroblast activation protein (Fap), hypoxanthine phosphoribosyltransferase-1 (Hprt1), platelet-derived growth factor subunit B (Pdgfb), phosphoinositide-3-kinase regulatory subunit-1 (Pik3r1), and vascular cell adhesion molecule-1 (Vcam1) qPCR primer with qPCR and electrophoresis validation. The procedure used in this approach was in silico analysis of primer from published articles and newly designed primer. The analysis was done with Primer-BLAST for gene specificity, Primer-Dimer, OligoCalc for hairpin formation, BLAST Nucleotide for identical sequence screening, and Clustal Omega for product length validation. Experimental validation was done using qPCR for optimal annealing temperature, priming ability, and amplificon specificity, and electrophoresis for product length validation. This assessment resulted in in silico and laboratory experimental validation of Acta2, Fap, Hprt1, Pdgfb, Pik3r1, and Vcam1 primer pairs producing suitable amplicon for qPCR using Rattus norvegicus cDNA with SYBR annealing temperature range of 60-65°C with three mM MgCl2. The primer pair can be used for further qPCR analysis under similar conditions and the procedure stated can be used as starting point for qPCR Primer preparation.

Keywords


qPCR primer; in silico; experimental validation

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References

Ahmed, F.E. et al., 2017. Role of Melt Curve Analysis in Interpretation of Nutrigenomics’ MicroRNA Expression Data. Cancer Genomics and Proteomics, 14(6), pp.469–481. doi: 10.21873/cgp.20057.

Alonso, G.C. et al., 2018. A Quest to Find Good Primers for Gene Expression Analysis of Candida albicans from Clinical Samples. Journal of Microbiological Methods, 147, pp.1–13. doi: 10.1016/j.mimet.2018.02.010.

Bourzac, K.M., LaVine, L.J. & Rice, M., 2003. Analysis of DAPI and SYBR Green I as Alternatives to Ethidium Bromide for Nucleic Acid Staining in Agarose Gel Electrophoresis. Journal of Chemical Education, 80(11), pp.1292–1296. doi: 10.1021/ed080p1292.

Brodin, J. et al., 2013. A Multiple-Alignment based Primer Design Algorithm for Genetically Highly Variable DNA Targets. BMC Bioinformatics, 14, 255. doi: 10.1186/1471-2105-14-255.

Brown, J.R. et al., 2021. Comparison of SARS-CoV-2 N Gene Real-time RT-PCR Targets and Commercially Available Mastermixes. Journal of Virological Methods, 295, 114215. doi: 10.1016/j.jviromet.2021.114215.

Cai, W. et al., 2019. MicroRNA-24 Attenuates Vascular Remodeling in Diabetic Rats through PI3K/Akt Signaling Pathway. Nutrition, Metabolism and Cardiovascular Diseases, 29(6), pp.621–632. doi: 10.1016/j.numecd.2019.03.002.

Camorani, S., Hill, B.S. & Fontanella, R., et al. 2017. Inhibition of BM-MSCs Homing Towards TNBC Microenvironment Using an Anti-PDGFRβ Aptamer. Theranostics, 7(14), pp.3595–3607

Canh, V.D. et al., 2020. Application of Capsid Integrity (RT-)qPCR to Assessing Occurrence of Intact Viruses in Surface Water and Tap Water in Japan. Water Research, 189, 116674. doi: 10.1016/j.watres.2020.116674.

Caselli, E. et al., 2021. Looking for More Reliable Biomarkers in Breast Cancer: Comparison Between Routine Methods and RT-qPCR. PLoS ONE, 16(9), e0255580. doi: 10.1371/journal.pone.0255580.

Cheung, H.W. et al., 2021. A Duplex qPCR Assay for Human Erythropoietin (EPO) Transgene to Control Gene Doping in Horses. Drug Testing and Analysis, 13(1), pp.113–121. doi: 10.1002/dta.2907.

Costa, A. et al., 2018. Fibroblast Heterogeneity and Immunosuppressive Environment in Human BC. Cancer Cell, 33(3), pp.463-479.e10.

Covey, S.D., 2021. An Adaptable Dry Lab for SYBR Based RT-qPCR Primer Design to Reinforce Concepts in Molecular Biology and Nucleic Acids. Biochemistry and Molecular Biology Education, 49(2), pp.262–270. doi: 10.1002/bmb.21446.

Cuevas-Ferrando, E. et al., 2021. Platinum Chloride-based Viability RT-qPCR for SARS-CoV-2 Detection in Complex Samples. Scientific Reports, 11, 18120. doi: 10.1038/s41598-021-97700-x.

D’haene, B., Vandesompele, J. & Hellemans, J., 2010. Accurate and Objective Copy Number Profiling using Real-Time Quantitative PCR. Methods, 50(4), pp.262–270. doi: 10.1016/j.ymeth.2009.12.007.

Dragan, A.I. et al., 2012. SYBR Green I: Fluorescence Properties and Interaction with DNA. Journal of Fluorescence, 22(4), pp.1189–1199. doi: 10.1007/s10895-012-1059-8.

Ferreira, I.D., do Rosario, V.E. & Cravo, P.V.L., 2006. Real-time Quantitative PCR with SYBR Green I Detection for Estimating Copy Numbers of Nine Drug Resistance Candidate Genes in Plasmodium falciparum. Malaria Journal, 5(1). doi: 10.1186/1475-2875-5-1.

Fiechter, R.H. et al., 2021. IL-12p40/IL-23p40 Blockade with Ustekinumab Decreases the Synovial Inflammatory Infiltrate through Modulation of Multiple Signaling Pathways Including MAPK-ERK and Wnt. Frontiers in Immunology, 12, 611656. doi: 10.3389/fimmu.2021.611656.

Fittipaldi, M., Codony, F. & Morato, J., 2010. Comparison of Conventional Culture and Real-Time Quantitative PCR using SYBR Green for Detection of Legionella pneumophila in Water Samples. African Journals Online, 36(4), pp.417–424. doi: 10.4314/wsa.v36i4.58411.

Gaby, J.C. & Buckley, D.H., 2017. The Use of Degenerate Primers in qPCR Analysis of Functional Genes Can Cause Dramatic Quantification Bias as Revealed by Investigation of nifH Primer Performance. Microbial Ecology, 74(3), pp.701–708. doi: 10.1007/s00248-017-0968-0.

Goda, T., Tabata, M. & Miyahara, Y., 2015. Electrical and Electrochemical Monitoring of Nucleic Acid Amplification. Frontiers in Bioengineering and Biotechnology, 3(29). doi: 10.3389/fbioe.2015.00029.

Gregory, S.M. et al., 2012. Latent Kaposi’s Sarcoma-Associated Herpesvirus Infection of Monocytes Downregulates Expression of Adaptive Immune Response Costimulatory Receptors and Proinflammatory Cytokines. Journal of Virology, 86(7), pp.3916–3923. doi: 10.1128/jvi.06437-11.

Hargreaves, S.K., Roberto, A.A. & Hofmockel, K.S., 2013. Reaction- and Sample-specific Inhibition Affect Standardization of qPCR Assays of Soil Bacterial Communities. Soil Biology and Biochemistry, 59, pp.89–97. doi: 10.1016/j.soilbio.2013.01.007.

Hashish, A. et al., 2021. Development and Validation of Two Diagnostic Real-Time PCR (Taqman) Assays for the Detection of Bordetella avium from Clinical Samples and Comparison to the Currently Available Real-Time Taqman PCR Assay. Microorganisms, 9, 2232. doi: 10.3390/microorganisms9112232.

Hu, Y. et al., 2012. Glioma Cells Promote the Expression of VCAM-1 on BM-MSCs: a Possible Mechanism for Their Tropism Toward Gliomas. Journal of Molecular Neuroscience. 48(1), pp.127–135.

Jiménez, C. et al., 2000. Role of the PI3K Regulatory Subunit in the Control of Actin Organization and Cell Migration. The Journal of Cell Biology, 151(2), pp.249–261.

Johnston, A.D. et al., 2019. PrimerROC: Accurate Condition-Independent Dimer Prediction using ROC Analysis. Scientific Reports, 9, 209. doi: 10.1038/s41598-018-36612-9.

Kang, S.J. et al., 2021. Comparison of Seven Commercial TaqMan Master Mixes and Two Real-Time PCR Platforms Regarding the Rapid Detection of Porcine DNA. Food Science of Animal Resources, 41(1), pp.85–94. doi: 10.5851/KOSFA.2020.E80.

Landry, N.M., Rattan, S.G. & Dixon, I.M.C., 2019. An Improved Method of Maintaining Primary Murine Cardiac Fibroblasts in Two-Dimensional Cell Culture. Scientific Reports, 9, 12889. doi: 10.1038/s41598-019-49285-9.

Langlois, V.S. et al., 2021. The Need for Robust qPCR-based eDNA Detection Assays in Environmental Monitoring and Species Inventories. Environmental DNA, 3(3), pp.519–527. doi: 10.1002/edn3.164.

Lee, S.B. et al., 2016. Ethyl Acetate Fraction of Amomum xanthioides Exerts Antihepatofibrotic Actions via the Regulation of Fibrogenic Cytokines in a Dimethylnitrosamine-Induced Rat Model. Evidence-based Complementary and Alternative Medicine, 2016, 6014380. doi: 10.1155/2016/6014380.

Luna, G.M. et al., 2012. A New Molecular Approach based on qPCR for the Quantification of Fecal Bacteria in Contaminated Marine Sediments. Journal of Biotechnology, 157(4), pp.446–453. doi: 10.1016/j.jbiotec.2011.07.033.

Morinha, F., Magalhães, P. & Blanco, G., 2020. Different qPCR Master Mixes Influence Telomere Primer Binding within and between Bird Species. Journal of Avian Biology, 51(2), e02352. doi: 10.1111/jav.02352.

Nakano, S.I. et al., 1999. Nucleic Acid Duplex Stability: Influence of Base Composition on Cation Effects. Nucleic Acids Research, 27(14), pp.2957–2965. doi: 10.1093/nar/27.14.2957.

Ouldridge, T.E. et al., 2013. DNA Hybridization Kinetics: Zippering, Internal Displacement and Sequence Dependence. Nucleic Acids Research, 41(19), pp.8886–8895. doi: 10.1093/nar/gkt687.

Petrovan, V. et al., 2020. Evaluation of Commercial qPCR Kits for Detection of SARS-CoV-2 in Pooled Samples. Diagnostics, 10(7), 472. doi: 10.3390/diagnostics10070472.

Ririe, K.M., Rasmussen, R.P. & Wittwer, C.T., 1997. Product Differentiation by Analysis of DNA Melting Curves during the Polymerase Chain Reaction. Analytical Biochemistry, 245(2), pp.154–160. doi: 10.1006/abio.1996.9916.

Sato, H. et al., 2021. Downregulation of Mitochondrial Biogenesis by Virus Infection Triggers Antiviral Responses by Cyclic GMP-AMP Synthase. PLoS Pathogens, 17(10), p.e1009841. doi: 10.1371/journal.ppat.1009841.

Sharma, A. et al., 2021. Comparative Transcriptomic and Molecular Pathway Analyses of HL-CZ Human Pro-Monocytic Cells Expressing SARS-CoV-2 Spike S1, S2, NP, NSP15 and NSP16 Genes. Microorganisms, 9(6), p.1193. doi: 10.3390/microorganisms9061193.

Singh, V.K. et al., 2000. The Effect of Hairpin Structure on PCR Amplification Efficiency. Molecular Biology Today, 1(3), pp.67–69.

Su, Q., Sena-Esteves, M. & Gao, G., 2020. Titration of Recombinant Adeno-Associated Virus (rAAV) Genome Copy Number Using Real-Time Quantitative Polymerase Chain Reaction (qPCR). Cold Spring Harbor Protocols, 2020(5), pp.158–161. doi: 10.1101/pdb.prot095646.

Tajima, K. et al., 2001. Diet-Dependent Shifts in the Bacterial Population of the Rumen Revealed with Real-Time PCR. Applied and Environmental Microbiology, 67(6), pp.2766–2774. doi: 10.1128/AEM.67.6.2766-2774.2001.

Taylor, S. et al., 2010. A Practical Approach to RT-qPCR-Publishing Data that Conform to the MIQE Guidelines. Methods, 50(4), pp.S1–S5. doi: 10.1016/j.ymeth.2010.01.005.

Thornton, B. & Basu, C., 2011. Real-Time PCR (qPCR) Primer Design Using Free Online Software Received. Biochemistry and Molecular Biology Education, 39(2), pp.145–154. doi: 10.1002/bmb.20461.

Trigo, B.B. et al., 2021. In Silico and In Vitro Evaluation of Primers for Molecular Differentiation of Leishmania Species. Brazilian Journal of Veterinary Parasitology, 30(1), e022020. doi: 10.1590/s1984-296120201078.

Vanneste, K. et al., 2018. Application of Whole Genome Data for In Silico Evaluation of Primers and Probes Routinely Employed for the Detection of Viral Species by RT-qPCR using Dengue Virus as a Case Study. BMC Bioinformatics, 19(1), 312. doi: 10.1186/s12859-018-2313-0.

Vogels, C.B.F. et al., 2020. Analytical Sensitivity and Efficiency Comparisons of SARS-CoV-2 RT–qPCR Primer–Probe Sets. Nature Microbiology, 5(10), pp.1299–1305. doi: 10.1038/s41564-020-0761-6.

Von Ahsen, N., Wittwer, C.T. & Schütz, E., 2001. Oligonucleotide Melting Temperatures under PCR Conditions: Nearest-Neighbor Corrections for Mg2⫹, Deoxynucleotide Triphosphate, and Dimethyl Sulfoxide Concentrations with Comparison to Alternative Empirical Formulas. Clinical Chemistry, 47(11), pp.1956–1961. doi: 10.1093/clinchem/47.11.1956.

Vulchi, R., Daane, K.M. & Wenger, J.A., 2021. Development of DNA Melt Curve Analysis for the Identification of Lepidopteran Pests in Almonds and Pistachios. Insects, 12(6), 553. doi: 10.3390/insects12060553.

Wang, H. et al., 2019. An Exonuclease-assisted Fluorescence Sensor for Assaying Alkaline Phosphatase based on SYBR Green I. Molecular and Cellular Probes, 45, pp.26–30. doi: 10.1016/j.mcp.2019.04.002.

Yang, T., Liu, J. & Chen, J., 2020. Compared with Conventional PCR Assay, qPCR Assay Greatly Improves the Detection Efficiency of Predation. Ecology and Evolution, 10(14), pp.7713–7722. doi: 10.1002/ece3.6494.

Ye, J. et al., 2012. Primer-BLAST: A Tool to Design Target-Specific Primers for Polymerase Chain Reaction. BMC Bioinformatics, 13, 134. doi: 10.1186/1471-2105-13-134.



DOI: https://doi.org/10.22146/jtbb.71765

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