Quantitative PCR of Small Nucleic Acids: Size Matters

Quantitative dysregulation in small nucleic acids (NA), such as microRNA (miRNA), extracted from minimally invasive biopsies, such as, blood, stool, urine, nose, throat, are promising biomarker for diseases diagnosis and management. We quantify the effect of the extra step of poly(A) ligation for cDNA synthesis and small size of the NA on the limit of quantification (LOQ) of quantitative PCR (qPCR), the gold standard to measure copy number. It was discovered that for small NA, the cycle threshold, Ct that is proportional to -log[c], where [c] is the concentration of the target NA exhibits a sharp transition. The results indicate that although the limit of detection (LOD) of qPCR can be in femtomolar range, the LOQ is significantly reduced by well over three orders of magnitude, in picomolar range. Specifically, the study reveals that the PCR product length is the primary reason the limitation on LOQ and is explicitly shown to be an important consideration for primer design for qPCR in general.