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Laboratory Methods for Investigating Nuclear and Cytoplasmic Genomes and Transcriptome

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Abstract

A cell usually has several categories of DNA. For example, the widely used Agrobacterium tumefaciens system used for plant genetic transformation has a large plasmid called a tumor-inducing (Ti) plasmid or helper Ti plasmid and a much smaller binary vector plasmid. A plant cell usually has nuclear, plastidic (chloroplast, amyloplast, and others), and mitochondrial genomes. Depending on the research objectives, such as organelle genome sequencing, it is often necessary to extract DNA and RNA separately from these different organelles or DNA macromolecules. Various laboratory approaches are also often required for validating microarray or second-generation sequencing results. This chapter describes the following 11 techniques: (1) in situ hybridization for studying tissue-specific gene expression, (2) mitochondrial visualization using rhodamine staining and confocal microscopy, (3) using polarized microscope in observing starch granules without staining, (4) differential preparation of Agrobacterium Ti plasmid and binary plasmid using a noncommercial kit, (5) isolation of nuclei for DNA preparation, (6) chloroplast DNA extraction, (7) mitochondrial DNA extraction, (8) total DNA/RNA preparation, (9) enriched mitochondrial RNA preparation, (10) high-resolution DNA melting analysis for studying gene expression, and (11) transcriptome electrophoretic fingerprinting. Most of the protocols are directly usable. If a protocol is very long such as the in situ hybridization protocol or highly dependent on a manual for specialized equipment such as the confocal microscope, the chapter provides a general description of the methods and discusses the aspects critical for resolution. In addition to their use in somatic genome research, these protocols can be useful in training students and new researchers in basic molecular biology techniques.

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Acknowledgments

The author thanks Mr. Muhammad Haroon of Agriculture and Agri-Food Canada for verification of the agrobacterial prep methods and Professor Danielle Donnelly of McGill University for editing the chapter. Part of the work was conducted in the Laboratoire d’Amélioration des Plantes (Professeur Yves Demarly) de la Université de Paris-Sud-XI, the Laboratoire de Génétique Moléculaire des Plantes (Dr. Fernand Vedel) du Centre National de la Recherche Scientifique de France (name of lab head), the Departments of Botany and Plant Pathology (Professor Thomas K. Hodges) and Biological Sciences (Professor Stanton B. Gelvin) of Purdue University, and the Department of Biology of McGill University (Professor Gregory G. Brown). All the remaining parts were conducted in the author’s laboratory (Cell Biotechnology) at the Department of Biology of Peking University and the authors’ laboratory (Molecular Genetics) at the Potato Research Centre of Agriculture and Agri-Food Canada, Fredericton.

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  1. Potato Research Centre, Agriculture and Agri-Food Canada, 850 Lincoln Road, P.O. Box 20280, Fredericton, NB E3B 4Z7, Canada

    Prof. Dr. Xiu-Qing Li

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  1. Prof. Dr. Xiu-Qing Li
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Editors and Affiliations

  1. Potato Research Centre, Agriculture and Agri-Food Canada, Government of Canada, Fredericton, New Brunswick, Canada

    Xiu-Qing Li

  2. Plant Science Department, McGill University, Sainte-Anne-de-Bellevue, Québec, Canada

    Danielle J. Donnelly

  3. Department of Biomedicine, Aarhus University, Herning, Denmark

    Thomas G. Jensen

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Li, XQ. (2015). Laboratory Methods for Investigating Nuclear and Cytoplasmic Genomes and Transcriptome. In: Li, XQ., Donnelly, D., Jensen, T. (eds) Somatic Genome Manipulation. Springer, New York, NY. https://doi.org/10.1007/978-1-4939-2389-2_14

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