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

Abstract

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.

Liquid lncRNA Biopsy for the Evaluation of Locally Advanced and Metastatic Squamous Cell Carcinomas of the Head and Neck

“…In the study, the 90 lncRNAs potentially connected with cancer and well-annotated in the lncRNA database (www.lncrnadb.org) were analyzed using the commercially available LncProfiler qPCR Array Kit (System Biosciences). The reaction consisted of three steps: (i) poly-A tailing, (ii) annealing anchor dT adaptor, (iii) complementary cDNA synthesis [42]. For the poly-A tailing step, 5 µL of RNA (about 100 ng) was used and mixed with 2 µL 5x PolyA Buffer, 1 µL MnCl 2 , 1.5 µL ATP and 0.5 µL polyA polymerase and incubated for 30 min at a temperature of 37 • C. Next, 0.5 µL of Oligo(dT) adapter was added and the reaction was heated for 5 min at 60 • C, and later cooled to room temperature.…”

The role of NEAT1 lncRNA in squamous cell carcinoma of the head and neck is still difficult to define

“…Patients’ RNA samples, tumour and matched adjacent normal, were taken from a previous study [ 31 ]. Expression levels of NEAT1 (family) in cell lines and patients’ samples were measured using lncProfiler qPCR Array Kit (SBI) and SYBR Green 2x Master Mix (Roche) as described previously [ 32 ]. All real-time PCR data were analysed by calculating the 2-ΔCT, normalising against the mean of reference genes (18S rRNA, RNU43, GAPDH, LAMIN A/C, U6) from the quantification plate.…”

The Long and the Short of It: NEAT1 and Cancer Cell Metabolism

“…Quantifying the relative abundance of NEAT1 isoforms is not straightforward. Kolenda et al [ 119 ] compared cDNA synthesis protocols for various cancer-associated lncRNAs; however, isoform differentiation was not a primary outcome. Similarly, the RNA purification method used can significantly influence the relative abundance of isoforms, with a heating step liberating NEAT1_2 from paraspeckle complexes and increasing yields [ 120 ].…”