Challenges for Accurate Quantification of RNA

Ribonucleic acid (RNA) quantification is an essential technique in biology. There has been remarkable progress in RNA quantification techniques over the recent years; however, the specificity of these techniques to quantify a very small amount of RNA is doubtful because of factors which can inhibit precise quantification. To develop a technique that leads to the most sensitive RNA quantification, these problems must be overcome. In this article, we first review the factors that inhibit precise quantification of RNA: the quality of RNA, secondary structure of RNA, efficiency of the enzyme reaction, annealing conditions, limitations of the experimental protocol and equipment, and detection sensitivity of the equipment. Next, we discuss the possible methods which contribute to these factors: RNA quality control focused on target RNA degradation, isothermal amplification, techniques for avoiding amplification errors, RNase H-dependent PCR, targeting using a fluorescent-labeled probe, targeting using a padlock probe, bridged/locked nucleic acid (BNA/LNA) and peptide nucleic acid (PNA), and the clustered regularly interspaced short palindromic repeat (CRISPR) system. One of the goals for the development of an ultrasensitive RNA quantification technique is the absolute quantification of RNA. Here, we discuss the techniques used for this type of RNA quantification.