Journal: International Journal of Molecular Sciences
Article Title: Evaluation and Adaptation of a Laboratory-Based cDNA Library Preparation Protocol for Retrospective Sequencing of Archived MicroRNAs from up to 35-Year-Old Clinical FFPE Specimens
Figure Lengend Snippet: 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.
Article Snippet: The cDNA libraries were then sequenced (single-read 50 cycles) on a HiSeq 2500 Sequencing System (SY-401-2501, Illumina, San Diego, CA, USA).
Techniques: cDNA Library Assay, Formalin-fixed Paraffin-Embedded, Purification, Polyacrylamide Gel Electrophoresis, Staining, Polymerase Chain Reaction, Amplification, Agarose Gel Electrophoresis, Sequencing