Combining asymmetric PCR-based enzymatic amplification with silicon photonic microring resonators for the detection of lncRNAs from low input human RNA samples

Abstract

A method for quantifying biologically relevant long-non-coding RNAs by combining nucleic acid amplification via asymmetric polymerase chain reaction (PCR) with label-free PCR product detection using silicon photonic microring resonator arrays is described. This approach eliminates the need for fluorophores, which presents a limit for spectral multiplexing in conventional qPCR methods, and rather offers potential for much higher levels of plexity by spatially arraying capture probes. Here, we demonstrate the potential of this technique to detect two differentially expressed lncRNA transcripts and an internal control mRNA transcript in different commercial human tissue specimens, as well as in a glioblastoma cell line using only nanogram input amounts of total RNA. The obtained results were validated using single-plex RT-qPCR and found to be in good agreement, demonstrating the potential of this technique for lncRNA quantification applications.

Long noncoding RNAs: from genomic junk to rising stars in the early detection of cancer

“…However, to facilitate surface-hybridization process, an excess of one primer can be used so that after consuming the limiting primer, single-stranded products are linearly generated. This strategy, denoted as RT-asymmetric PCR, has been recently implemented to assess differential expression of two human long noncoding RNAs in a glioblastoma cell line and healthy brain tissues, finding a good agreement with qRT-PCR results [28], which are typically taken as the reference for the validation.…”

“…However, expression levels of housekeeping genes can vary in given situations that have motivated a lack of consensus on the use of internal standards for relative quantification of circulating transcriptome; consequently, their practical utility should be evaluated in each case. The endogenous controls typically used to monitor the expression profile of lncRNAs are β-actin[28] and glyceraldehyde-3-phosphate dehydrogenase (GAPDH)[31].…”

Advances in Electrochemical (Bio)Sensing Targeting Epigenetic Modifications of Nucleic Acids

“…Therefore, there is an opportunity for the emergence of new biosensing technologies to overcome these shortcomings and better meet the needs of POC disease evaluation . Indeed, the methods to detect epimarks or aberrant ncRNAs expression are evolving rapidly to offer improvements particularly with respect to the development of multiplexed, low cost and easy to operate bioanalytical platforms for near‐patient applications .…”

“…In addition, the susceptibility to contamination and limitation of using in centralized settings hinder their application to routine, on site determinations. RNA sequencing, although extensively used for lncRNA discovery and expression profiling, requires extensive bioinformatic data analysis and is still prohibitively expensive for studies involving a large number of clinical samples [9,26].…”

“…Numerous recent reports show that ncRNAs function as epigenetic regulators by modulating the expression of genes through transcriptional and post‐transcriptional mechanisms. The ncRNAs are broadly categorized into two major groups based on their length and using the arbitrary cut‐off of 200 nucleotides: small ncRNAs, which include Piwi‐interacting RNAs, small‐interfering RNAs and miRNAs; and lncRNAs . MiRNAs and lncRNAs are considered nowadays the most relevant ncRNAs in epigenetics.…”