Making translational transcriptomics SMARTer
Many samples used for RNA-seq during translational research are extraordinarily precious. Clinical samples such as circulating tumor cells and tissue biopsies may be limited to a few cells, or even a single cell. For studies in which repeated blood sampling is not possible, specimens may be available in finite quantities. And RNA from material such as FFPE or laser capture microdissection (LCM) samples may have low RIN values. When there are no second chances to obtain data, experimental methodology is crucial.
To enable transcriptional analysis in a variety of biomedical applications—including those where input RNA is rare or compromised—we offer kits using SMART cDNA synthesis technology for RNA-seq on Illumina, Ion Torrent, or Fluidigm C1 platforms. These include products for RNA-seq library generation and preparation from as little as 10 pg RNA, for use with degraded RNA, and for rRNA depletion.
SMART technology enables synthesis of full-length cDNA by leveraging the terminal transferase activity and template-switching ability of Moloney Murine Leukemia Virus Reverse Transcriptase (MMLV RT) (Figure 1). Full-length cDNA is used for amplification and validation, fragmentation, and library preparation for sequencing on Illumina HiSeq, MiSeq, or Genome Analyzer systems, or the Ion Torrent platform. A number of translational medicine transcriptome profiling studies have taken advantage of the powerful sensitivity and efficient workflows enabled by these kits.
Figure 1. SMARTer cDNA synthesis. When the SmartScribe RT reaches the 5' end of the RNA template, it adds a few additional nucleotides to the 3' end of the cDNA. A carefully designed SMARTer IIA oligo base-pairs with these additional nucleotides, creating an extended template. The enzyme then switches templates, continuing transcription until it reaches the end of the oligonucleotide. The resulting full-length cDNA contains the complete 5' end of the mRNA plus an anchor sequence that serves as a universal priming site for second strand synthesis and amplification by LD-PCR.