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Pseudogenes pp 103–115Cite as

Methods for Detecting Transcribed Pseudogenes: PCR on Regions of High Sequence Similarity Followed by Cloning and Sequencing

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Part of the book series: Methods in Molecular Biology ((MIMB,volume 1167))

Abstract

Identifying pseudogene transcription is problematic in many cases due to the high sequence similarity between pseudogenes and their parental genes. In this chapter, we detail the procedure for the detection of pseudogene transcription using the reverse transcription polymerase chain reaction (RT-PCR) method. The protocol comprises (1) extraction of total RNA, (2) first-strand cDNA synthesis from total RNA, (3) amplification of the cDNA by PCR, and (4) cloning and sequencing of the PCR products. Technical and practical guidance is provided, and the critical points during each of the steps are discussed. In particular, the importance of designing high specific PCR primers and thoroughly eliminating genomic DNA contamination from RNA preparation is emphasized.

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Acknowledgments

This work was supported by the National Natural Science Foundation of China (No. 81272429, to Dr. W. Ding) and the National Basic Research Program of China (973 Program, No. 2011CB965001, to Professor J. Dai and Dr. W. Ding).

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Authors and Affiliations

  1. Department of Biochemistry, Dalian Medical University, Dalian, China

    Wenyong Ding

  2. State Key Laboratory of Molecular Developmental Biology, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing, China

    Jianwu Dai Ph.D.

  3. Stem Cell and Regenerative Medicine Laboratory, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, #3 Zhongguancun Nanyitiao, Haidian District, Beijing, China

    Jianwu Dai Ph.D.

Authors
  1. Wenyong Ding
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  2. Jianwu Dai Ph.D.
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Corresponding author

Correspondence to Jianwu Dai Ph.D. .

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Editors and Affiliations

  1. Istituto Toscano Tumori, Pisa, Italy

    Laura Poliseno

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Ding, W., Dai, J. (2014). Methods for Detecting Transcribed Pseudogenes: PCR on Regions of High Sequence Similarity Followed by Cloning and Sequencing. In: Poliseno, L. (eds) Pseudogenes. Methods in Molecular Biology, vol 1167. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-0835-6_8

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  • DOI: https://doi.org/10.1007/978-1-4939-0835-6_8

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-0834-9

  • Online ISBN: 978-1-4939-0835-6

  • eBook Packages: Springer Protocols

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