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. 2021 Jun 25;11(7):1160.
doi: 10.3390/diagnostics11071160.

The Effect of Genomic DNA Contamination on the Detection of Circulating Long Non-Coding RNAs: The Paradigm of MALAT1

Athina N Markou  1 , Stavroula Smilkou  1 , Emilia Tsaroucha  2 , Evi Lianidou  1
Affiliations

The Effect of Genomic DNA Contamination on the Detection of Circulating Long Non-Coding RNAs: The Paradigm of MALAT1

Athina N Markou et al. Diagnostics (Basel). .

Abstract

The presence of contaminating gDNA in RNA preparations is a frequent cause of false positives in RT-PCR-based analysis. However, in some cases, this cannot be avoided, especially when there are no exons-intron junctions in the lncRNA sequences. Due to the lack of exons in few of long noncoding RNAs (lncRNAs) and the lack of DNAse treatment step in most studies reported so far, serious questions are raised about the specificity of lncRNA detection and the potential of reporting false-positive results. We hypothesized that minute amounts of gDNA usually co-extracted with RNA could give false-positive signals since primers would specifically bind to gDNA due to the lack of junction. In the current study, we evaluated the effect of gDNA and other forms of DNA like extrachromosomal circular DNAs (eccDNAs) contamination and the importance of including a DNAse treatment step on lncRNAsexpression.As a model, we have chosen as one of the most widely studied lncRNAs in cancer namely MALAT1, which lacks exons. When we tested this hypothesis in plasma and primary tissue samples from NSCLC patients, our findings clearly indicated that results on MALAT1 expression are highly affected by the presence of DNA contamination and that the DNAse treatment step is absolutely necessary to avoid false positive results.

Keywords: DNAse treatment; MALAT1; cfRNA; false positive results; gDNA; non-long coding RNA; tumor biomarkers.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Graphic summary of lncRNA MALAT1 reference sequence, MALAT1 gDNA and position of different pairs of primers designed used in various studies. The last pair (green) was the one designed in the present study.
Figure 2
Figure 2
B2M and MALAT1 expression before and after DNase treatment.
Figure 3
Figure 3
Outline of the experimental procedure.
Figure 4
Figure 4
(a): Effect of enzyme incubation time. (b):Effect of gDNA concentration.
Figure 4
Figure 4
(a): Effect of enzyme incubation time. (b):Effect of gDNA concentration.
See this image and copyright information in PMC

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