Optimization of small-scale sample preparation for high-throughput OpenArray analysis
Main Article Content
Keywords
Cells-to-CT, OpenArray, qPCR, RNeasy
Abstract
OpenArray is one of the most high-throughput qPCR platforms available but its efficiency can be limited by sample preparation methods that are slow and costly. To optimize the sample workflow for high-throughput qPCR processing by OpenArray, small-scale sample preparation methods were compared for compatibility with this system to build confidence in a method that maintains quality and accuracy while using less starting material and saving time and money. This study is the first to show that the Cells-to-CT kit can be used to prepare samples within the dynamic range of OpenArray directly from cultured cells in a single well of a 96-well plate when used together with a cDNA preamplification PCR step. Use of Cells-to-CT produced results of similar quality and accuracy to that of a preparation method using purified RNA in less than half the sample preparation time. While Cells-to-CT samples also exhibited slightly increased variance, which affects the ability of OpenArray to distinguish small differences in gene expression, overall gene expression mean results correlated well between small-scale methods. This work demonstrates that Cells-to-CT with preamplification can be used to reliably prepare samples for OpenArray analysis while saving time, money, and starting material.
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References
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2. Broccanello C, Gerace L, Stevanato P (2020) QuantStudio™ 12K Flex OpenArray® System as a Tool for High-Throughput Genotyping and Gene Expression Analysis. Methods Mol Biol 2065:199–208.
3. Prokopec SD, Watson JD, Waggott DM, Smith AB, Wu AH, et al. (2013) Systematic evaluation of medium-throughput mRNA abundance platforms. RNA 19(1):51–62.
4. Li P, Grigorenko E, Funari V, Enright E, Zhang H, et al. (2013) Evaluation of a high-throughput, microfluidics platform for performing TaqMan™ qPCR using formalin-fixed paraffin-embedded tumors. Bioanalysis: 5(13):1623–1633.
5. Patel SN, Wu Y, Bao Y, Mancebo R, Au-Young J, et al. (2013) TaqMan® OpenArray® high-throughput transcriptional analysis of human embryonic and induced pluripotent stem cells. Methods Mol Biol 997:191–201.
6. Dalgard CL, Polston KF, Sukumar G, Mallon CT, Wilkerson MD, et al. (2016) MicroRNA Expression Profiling of the Armed Forces Health Surveillance Branch Cohort for Identification of "Enviro-miRs" Associated With Deployment-Based Environmental Exposure. J Occup Environ Med 58(8 Suppl 1):S97–S103.
7. Life Technologies. (2012). Applied Biosystems™ QuantStudio™ 12K Flex Real-Time PCR System OpenArray® Experiments: User Guide. Retrieved from https://www.thermofisher.com/content/dam/LifeTech/migration/files/pcr/pdfs.par.36605.file.dat/4470935b.pdf
8. Forreryd A, Johansson H, Albrekt AS, Lindstedt M (2014) Evaluation of high throughput gene expression platforms using a genomic biomarker signature for prediction of skin sensitization. BMC Genomics 15(1):379.
9. Life Technologies. (2012). TaqMan® Gene Expression Cells-to-CT™ Kit: User Guide. Retrieved from https://assets.thermofisher.com/TFS-Assets/LSG/manuals/cms_056225.pdf
10. Abruzzese R, Fekete R, Kephart D (2010) Cells-to-CT Kits: Next Generation Gene Expression Analysis Workflows that Eliminate Sample Purification. J Lab Autom 15(5): 362–368.
11. Van Peer G, Mestdagh P, Vandesompele J (2012) Accurate RT-qPCR gene expression analysis on cell culture lysates. Sci Rep 2:222.
12. Fox BC, Devonshire AS, Baradez MO, Marshall D, Foy CA (2012) Comparison of reverse transcription-quantitative polymerase chain reaction methods and platforms for single cell gene expression analysis. Anal Biochem 427(2):178–186.
13. Wang W, Gao Q, Yang M, Zhang X, Yu L, et al. (2015) Up-regulation of lysosomal TRPML1 channels is essential for lysosomal adaptation to nutrient starvation. Proc Natl Acad Sci U S A 112(11):E1373–E1381.
14. Thermo Fisher Scientific. (2016). Crt, a relative threshold method for qPCR data analysis on the QuantStudio 12K Flex system with OpenArray technology: Application Note. Retrieved from http://tools.thermofisher.com/content/sfs/brochures/CO28730-Crt-Tech-note_FLR.pdf
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Published
Feb 1, 2021
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How to Cite
1.
Abraham NA, Campbell AC, Hirst WD, Nezich CL. Optimization of small-scale sample preparation for high-throughput OpenArray analysis. J Biol Methods [Internet]. 2021 Feb. 1 [cited 2024 Apr. 20];8(1):e143. Available from: https://jbmethods.org/index.php/jbm/article/view/339
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