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.
Highlights
Non-coding RNAs are RNA molecules that are not translated into a protein but their functions are undoubtedly crucial in several mechanisms
We evaluated the effectiveness of the DNAse treatment step by analyzing genomic DNA (gDNA) samples at concentrations of 20 ng/μL and 5 ng/μL, using 5 and 10 min as incubation time. gDNA was added in the same RNA samples that were split into two aliquots of 10 μL each
Discussion long noncoding RNAs (lncRNAs) have been evaluated as novel tumor biomarkers, in diagnosis and prognosis, and in targeted therapy for different types of cancer [40,41]
Summary
Non-coding RNAs (ncRNAs) are RNA molecules that are not translated into a protein but their functions are undoubtedly crucial in several mechanisms. They are divided into short ncRNAs and long ncRNAs(lncRNAs) based on their nucleotide length. The lncRNAs are non-protein-coding transcripts with a length over 200 nucleotides (nt), and consist of the broadest class of ncRNAs [1]. LncRNAs were found to be involved in many biological processes [3] and have gained considerable interest as principal regulators of gene expression in several different ways [4,5]. Numerous studies have shown that lncRNAs are abnormally expressed in many cancers, such as breast, lung and prostate [6,7]
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