Designed Primer_Based RNA Sequencing

DP-Seq amplifies mRNA from limited starting material, as low as 50 pg (Bhargava et al., 2014). In this method, a specific set of heptamer primers is designed. The enriched poly(A)-selected mRNA undergoes first-strand cDNA synthesis. Next, the designed primers are hybridized to the first-strand cDNA, followed by second-strand synthesis and PCR. Deep sequencing of the amplified DNA allows for accurate detection of specific mRNA expression at the single-cell level.


  • As little as 50 pg of starting material can be used
  • Little transcript-length bias


  • Sequences of the target areas must be known to design the heptamers
  • Exponential amplification during PCR can lead to primer-dimers and spurious PCR products (Bhargava et al., 2014)
  • Some read-length bias


Illumina Library prep and Array Kit Selector


Friedmann-Morvinski D., Bhargava V., Gupta S., Verma I. M. and Subramaniam S. Identification of therapeutic targets for glioblastoma by network analysis. Oncogene. 2015;

Kolodziejczyk A. A., Kim J. K., Svensson V., Marioni J. C. and Teichmann S. A. The Technology and Biology of Single-Cell RNA Sequencing. Mol Cell. 2015;58:610-620

Head S. R., Komori H. K., LaMere S. A., et al. Library construction for next-generation sequencing: overviews and challenges. Biotechniques. 2014;56:61-64, 66, 68, passim


Bhargava V., Head S. R., Ordoukhanian P., Mercola M. and Subramaniam S. Technical variations in low-input RNA-seq methodologies. Sci Rep. 2014;4:3678