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Reverse Transcription of 18S rRNA with Poly(dT)18 and Other Homopolymers

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Abstract

Ribosomal 18S RNA is widely used as a housekeeping gene in expression studies, including end-point PCR, Northern analysis, and real-time experiments. However, there are two disadvantages and two points of error introduction in using 18S rRNA as a reference gene. First, 18S has no poly(A) tail, so it is commonly reverse transcribed with specific primers or random hexamers, independently from poly(dT)-primed transcripts. Secondly, due to its abundance, the 18S cDNA must be extensively diluted to be comparable to the tested genes. In this study, 18S rRNA from five taxonomically diverse plant species, including Physcomitrella patens, Adiantum capillus-veneris, Centaurium erythraea, Arabidopsis thaliana, and Zea mays, was successfully reverse transcribed (RT) using poly(dT)18. As all other homopolymers, including poly(dA)18, poly(dC)18, and poly(dG)18, could serve as RT primers, it was concluded that homopolymers anneal by mispriming at the sites of complementary homopolymeric runs or segments rich in complementary base. Poly(dC)18 was the most efficient as RT primer, and the only one which interfered with subsequent PCR, giving species-specific pattern of products. Poly(dT)-primed RT reactions were less efficient in comparison to specific primer or random hexamer-primed reactions. Homopolymeric priming of 18S in RT reactions is general in terms of RNA origin and the method of RNA isolation and is possibly applicable to other tailless housekeeping genes.

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Abbreviations

RT:

Reverse transcription

F:

Forward

R:

Reverse

CT :

Threshold cycle

HP:

Homopolymers

Gln:

Glutamine synthetase

Ckx:

Cytokinin oxidase/dehydrogenase

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Acknowledgments

We thank Dr. Saša Malkov for assembled C. erythraea 18S sequence and Dr. Marko Sabovljević for providing initial moss cultures. This research was supported by the Ministry of Education and Science of the Republic of Serbia, contract no. OI 173024.

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Authors and Affiliations

  1. Institute for Biological Research “Siniša Stanković”, University of Belgrade, Bul. Despota Stefana 142, 11060, Belgrade, Serbia

    Milica D. Bogdanović, Milan B. Dragićević, Nikola T. Tanić, Slađana I. Todorović, Danijela M. Mišić, Suzana T. Živković & Ana D. Simonović

  2. Department of Cell and Metabolic Biology, Leibniz Institute of Plant Biochemistry, Weinberg 3, 06120, Halle (Saale), Germany

    Alain Tissier

Authors
  1. Milica D. Bogdanović
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  2. Milan B. Dragićević
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  3. Nikola T. Tanić
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  4. Slađana I. Todorović
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  5. Danijela M. Mišić
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  6. Suzana T. Živković
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  7. Alain Tissier
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  8. Ana D. Simonović
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Corresponding author

Correspondence to Milica D. Bogdanović.

Additional information

Milica D Bogdanović and Ana D Simonović contributed equally to this work.

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Bogdanović, M.D., Dragićević, M.B., Tanić, N.T. et al. Reverse Transcription of 18S rRNA with Poly(dT)18 and Other Homopolymers. Plant Mol Biol Rep 31, 55–63 (2013). https://doi.org/10.1007/s11105-012-0474-y

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  • DOI: https://doi.org/10.1007/s11105-012-0474-y

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