eISSN: 1896-9151
ISSN: 1734-1922
Archives of Medical Science
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abstract:
Basic research

Quantification of long non-coding RNAs using qRT-PCR: comparison of different cDNA synthesis methods and RNA stability

Tomasz Kolenda
,
Marcel Ryś
,
Kacper Guglas
,
Anna Teresiak
,
Renata Bliźniak
,
Jacek Mackiewicz
,
Katarzyna Lamperska

Arch Med Sci
Online publish date: 2019/01/30
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Introduction
Long non-coding RNAs (lncRNAs), a class of regulatory RNA molecules, are over 200 nucleotides long and could be used as a new potential biomarker, but their detection methods such as qRT-PCR are still not validated, and the influence of RNA degradation on lncRNA quantification is not clear. In this study, commercially available cDNA synthesis kits were tested and the influence of RNA degradation was compared.

Material and methods
Total RNA from FaDu cells was isolated and high quality RNA and highly degraded RNA samples were used. Reverse transcription was performed using three different commercially available kits and quantifications were performed using lncRNA Primer Plate and SYBR Green I Master by LightCycler 96. qRT-PCR was performed using three different cDNA samples and results are presented as the mean Ct values. A p-value < 0.05 was considered to be significant.

Results
Lower lncRNA Ct values (61/90; 67.78%) after qRT-PCR quantification were observed for cDNA synthesized using random hexamer primers preceded by polyA-tailing and adaptor-anchoring steps. It was observed that 9/90 (10.00%) lncRNAs were not detectable using different cDNA synthesis methods. For 75/90 (83%) lncRNAs, RNA degradation weakly influenced lncRNA Ct values and no differences were observed between high quality RNA and degraded samples. Seventy percent of examined lncRNAs showed significantly different Ct values depending on RNA degradation.

Conclusions
cDNA synthesis kits with random hexamer primers preceded by polyA-tailing and adaptor-anchoring steps allows enhancement of lncRNA quantification specificity and sensitivity. In most cases degradation of RNA samples does not affect lncRNA quantification because these molecules have good stability.

keywords:

lncRNA, cDNA synthesis, qRT-PCR, RNA stability and degradation

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