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Our understanding of transcriptome biology is undergoing a revolution that is revealing that the regulation of expression and the types and functions of RNA are far more complex than was previously thought. Enrichment of whole transcriptome RNA by depleting ribosomal RNA (rRNA) species using our RiboMinus™ technology has the potential to enhance discovery using gene expression microarrays, RNA-Seq, and other methods. The Ambion® WT Expression Kit for RNA amplification prior to microarray analysis circumvents the need to deplete rRNA by selectively eliminating rRNA from reverse transcription in the amplification process. Both of these technologies have the potential to facilitate our understanding of the new paradigm forming around the role of the transcriptome in both normal physiological and pathological processes.

RiboMinus™ Technology

 

Our RiboMinus™ technology utilizes specific locked nucleic acid (LNA®) capture probes to bind ribosomal RNA and subsequently remove it from the sample via binding to streptavidin-coated Dynabeads® magnetic beads. The remaining whole-transcriptome RiboMinus™ RNA is suitable for direct sequencing using any next-generation sequencing platforms or microarray analysis.

Ribosomal Binding Site Sequence Requirements

The sequence and structure of the 5' untranslated region (UTR) of mRNA transcripts plays a significant role in regulation of protein synthesis. In prokaryotes, the ribosome binding site (RBS), which promotes efficient and accurate translation of mRNA, is called the Shine-Dalgarno sequence after the scientists who first described it. This purine-rich 5' UTR sequence is complementary to the UCCU core sequence of the 3'-end of 16S rRNA (located within the 30S small ribosomal subunit). Various Shine-Dalgarno sequences have been found in prokaryotic mRNAs (see Figure 1 for the consensus sequence). These sequences lie about 10 nucleotides upstream from the AUG start codon. Activity of prokaryotic RBSs can be influenced by the length and nucleotide composition of the spacer separating the RBS and the initiator AUG.

 

 

 

Figure 1. Consensus RBS Sequences. The +1 A is the first base of the AUG initiator codon (shaded) responsible for binding of fMet-tRNAfMet. The underline indicates the ribosomal binding site sequence, which is required for efficient translation

 

 

In eukaryotes, the Kozak sequence, A/GCCACCAUGG, which lies within a short 5' UTR, helps direct translation of mRNA. mRNAs lacking the Kozak consensus sequence may be translated efficiently in in vitro translation systems if they possesses a moderately long 5' UTR that lacks stable secondary structure. Our data demonstrate that in contrast to the E. coli ribosome, which preferentially recognizes the Shine-Dalgarno sequence, eukaryotic ribosomes (such as those found in retic lysate used in in vitro translation systems) can efficiently use either the Shine-Dalgarno or the Kozak ribosomal binding sites.

Ribosomal RNA Sizes

SpeciesrRNASize (kb)
Human18S 1.9
28S 5
Mouse18S 1.9
28S 4.7
Drosophila 18S 2
28S 4.1*
Tobacco Leaf 16S 1.5
18S 1.9
23S 2.9
25S 3.7
Yeast 18S 2
26S 3.8
E. coli16S 1.5
23S 2.9
Xenopus18S 4
28S 1.8

*Drosophila 28S ribosomal RNA is processed into 2 fragments that migrate in a similar manner to the 18S rRNA.