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Article

An efficient method for high-fidelity messenger RNA amplification from a small amount of total RNA

  • Authors:
    • Mitsuru Chiba
    • Saya Asari
    • Misako Kimura
    • Toshiya Nakamura
  • View Affiliations / Copyright

    Affiliations: Department of Biomedical Sciences, Division of Medical Life Sciences, Hirosaki University Graduate School of Health Sciences, Hirosaki, Aomori 036-8564, Japan, Department of Medical Technology, Hirosaki University Graduate School of Health Sciences, Hirosaki, Aomori 036-8564, Japan
  • Pages: 105-110
    |
    Published online on: October 4, 2012
       https://doi.org/10.3892/br.2012.15
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Abstract

Comprehensive analyses of gene expression have been carried out by the development of microarrays and deep sequencers. However, it is difficult to obtain comprehensive information on gene expression from a small amount of ribonucleic acid (RNA). Therefore, we investigated the reproducibility and application of T7 RNA polymerase-mediated transcription, adaptor ligation and polymerase chain reaction (PCR) amplification, followed by T7 transcription (TALPAT), an efficient method for amplifying poly (A)-positive RNA, such as messenger RNA (mRNA). When amplified complementary RNA (cRNA) was electrophoresed, a large number of amplified cRNA was detected in the size of 0.2-0.5 kb. This indicates that the region up to 0.2-0.5 kb from the 3' end of the original mRNA was amplified by the TALPAT method. Seven housekeeping genes, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), hydroxymethylbilane synthase (HMBS), hypoxanthine phosphoribosyltransferase (HPRT1), ribosomal protein L13a (RPL13A), succinate dehydrogenase complex (SDHA), TATA box-binding protein (TBP) and ubiquitin C (UBC), showed high reproducibility (square of the correlation coefficient, R2=0.9954), according to scatter plots of Ct values obtained in the real-time PCR analysis of amplified cRNA. In addition, relative expression ratios of amplified cRNA of the seven housekeeping genes were approximately equal to the ratio of the original RNA solution. Furthermore, cRNA was amplified from 20 pg total RNA. In the present study, we confirmed the characteristics of mRNA amplification using the TALPAT method. This method may be applicable to mRNA and poly (A)-positive non-coding RNA amplification, using a small amount of RNA from single, laser-captured and sorted cells, as well as exosomes from serum, urine and body fluids.
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Copy and paste a formatted citation
Spandidos Publications style
Chiba M, Asari S, Kimura M and Nakamura T: An efficient method for high-fidelity messenger RNA amplification from a small amount of total RNA. Biomed Rep 1: 105-110, 2013.
APA
Chiba, M., Asari, S., Kimura, M., & Nakamura, T. (2013). An efficient method for high-fidelity messenger RNA amplification from a small amount of total RNA. Biomedical Reports, 1, 105-110. https://doi.org/10.3892/br.2012.15
MLA
Chiba, M., Asari, S., Kimura, M., Nakamura, T."An efficient method for high-fidelity messenger RNA amplification from a small amount of total RNA". Biomedical Reports 1.1 (2013): 105-110.
Chicago
Chiba, M., Asari, S., Kimura, M., Nakamura, T."An efficient method for high-fidelity messenger RNA amplification from a small amount of total RNA". Biomedical Reports 1, no. 1 (2013): 105-110. https://doi.org/10.3892/br.2012.15
Copy and paste a formatted citation
x
Spandidos Publications style
Chiba M, Asari S, Kimura M and Nakamura T: An efficient method for high-fidelity messenger RNA amplification from a small amount of total RNA. Biomed Rep 1: 105-110, 2013.
APA
Chiba, M., Asari, S., Kimura, M., & Nakamura, T. (2013). An efficient method for high-fidelity messenger RNA amplification from a small amount of total RNA. Biomedical Reports, 1, 105-110. https://doi.org/10.3892/br.2012.15
MLA
Chiba, M., Asari, S., Kimura, M., Nakamura, T."An efficient method for high-fidelity messenger RNA amplification from a small amount of total RNA". Biomedical Reports 1.1 (2013): 105-110.
Chicago
Chiba, M., Asari, S., Kimura, M., Nakamura, T."An efficient method for high-fidelity messenger RNA amplification from a small amount of total RNA". Biomedical Reports 1, no. 1 (2013): 105-110. https://doi.org/10.3892/br.2012.15
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