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. 2017 Mar 14;18(3):627. doi: 10.3390/ijms18030627

Figure 1.

Figure 1

Illustrated representation of the optimized barcoded cDNA library preparation procedure. Total RNA from formalin-fixed paraffin-embedded (FFPE) specimens (n = 18), are ligated with 3′ adapters (n = 18), purified on a 15% PAGE gel (using size markers (SM) RNA oligonucleotides) and excised after SYBR® gold staining of the gel. The combined purified products are ligated with a 5′ adapter, run on a 12% PAGE gel, excised and purified. The dual-adapter ligated small-RNA products are reverse transcribed, subjected to a pilot PCR for identification of adequate amplification cycle. A large-scale PCR is performed, and the products are separated on a 2% agarose gel, where the upper band (cDNA library) is purified and subjected to sequencing on an Illumina HiSeq-2500 sequencer.