Long noncoding RNAs (lncRNAs) play important roles in normal biology and disease, but studying them can be tricky. One key challenge is determining how many molecules of a particular lncRNA exist inside a cell. Traditional methods, such as single-molecule RNA fluorescence in situ hybridization (smFISH) and calibrated reverse-transcription quantitative PCR (RT-qPCR), have been used to estimate lncRNA levels. However, both approaches have limitations that can lead to inaccurate results.

Researchers from the University of North Carolina at Chapel Hill have developed a sequencing-based approach that provides a more reliable way to estimate lncRNA abundance. This method starts by determining the average amount of total RNA per cell. Then, RNA sequencing (RNA-seq) is performed using an rRNA-depletion protocol, with known amounts of spike-in control RNAs added. By comparing sequencing read counts to the spike-in RNAs, the researchers can estimate how many molecules of each transcript are present in a single cell.

Overview of sequencing-based approach to quantify lncRNA and mRNA intracellular abundance

This sequencing-based method has several advantages over traditional techniques. Unlike smFISH, which may underestimate RNA levels when molecules cluster in specific areas of the cell, RNA sequencing provides a broader and more quantitative view. Additionally, RT-qPCR can be affected by variations in reverse transcription efficiency, whereas the sequencing approach offers a more consistent measurement.

By improving the accuracy of lncRNA abundance measurements, this method could help researchers better understand how these molecules function in health and disease. It also allows scientists to study multiple lncRNAs and messenger RNAs (mRNAs) at once, making it a powerful tool for transcriptomics research.