Optimization of small-scale sample preparation for high-throughput OpenArray analysis

Authors

  • Neeta A. Abraham Biogen
  • Anne C. Campbell Biogen
  • Warren D. Hirst Biogen
  • Catherine L. Nezich Biogen

DOI:

https://doi.org/10.14440/jbm.2021.339

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.

References

Morrison T, Hurley J, Garcia J, Yoder K, Katz A, et al. (2006) Nanoliter high throughput quantitative PCR. Nucleic Acids Res 34(18):e123.

Broccanello C, Gerace L, Stevanato P (2020) QuantStudio

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Published

2021-02-01

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]. 2021Feb.1 [cited 2021Apr.12];8(1):e143. Available from: https://jbmethods.org/index.php/jbm/article/view/339

Issue

Section

Benchmarks