RNA-Mediated Oligonucleotide Annealing, Selection, and Ligation with Next-Generation Sequencing

RNA-mediated oligonucleotide annealing, selection, and ligation with next-generation sequencing (RASL-Seq) is a 2-dimensional RNA sequencing method to quantify expression profiles of several hundred genes, under thousands of different conditions (Li et al., 2012). Custom probe pairs are designed for each gene of interest. A pair of probes needs to contain: 1) One probe containing a common index primer on its 3′ end and a 20 nt oligonucleotide corresponding to the targeted exon sequence with a phosphate on the 5′ end; and 2) another probe with a P5 adapter on its 5′ end with a 20 nt sequence complementary to the exon that is adjacent to the other probe. The probe pairs are hybridized to the mRNA and separated from total RNA using oligo(dT)-biotin beads. A ligation step joins the probe pairs into a single PCR amplicon probe. The biotinylated mRNA strands are subsequently attached to streptavidin magnetic beads to elute the probe fragments. Next, P7 adapters are attached to the 3′ index primer, and the library undergoes PCR amplification before sequencing. The library is sequenced from the 40 nt ligated P5 primer, followed by sequencing from the P7 primer oligonucleotide.


  • Quantify genetic expression in large gene panels under thousands of different experimental conditions
  • Effective on low total RNA amounts (10 ng for about 1000 cells)
  • Can be performed on isolated RNA samples or cell lysates
  • Can be performed manually or automatically using a custom robot


  • Sequencing is carried out on short 40 nt fragments, which can cause problems in repetitive regions
  • High rate of random ligations can occur at low RNA levels
  • Automated process produces less robust results, due to random ligations during probe removal


Illumina Library prep and Array Kit Selector


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