For the last few years, stem cell researchers have been intrigued by miRNAs’ Houdini-esqe disappearing act as stem cells embrace their fate. Add in evidence showing how knocking out endogenous miRNA machinery like Ago2 can really add a few kinks to proper neural development and you get a stem cell community that’s fired up to take a deep dive into the miRNA repertoire involved in stem cell development.
Early on teams were quick to document the tangled web woven by miR-302 and miR-145 and their interactions with key stem cell regulators like Oct4 and Sox2, but as researcher’s appreciation for miRNA in stem cell regulation increases, so have the numbers of miRNAs that seem to be intricately involved in key stem cell processes.
A team of Garden State researchers at the Rutgers Stem Cell Research Center poured more fuel on the fire this week in PLoS One. Using ABI’s Sequencing by Oligo Ligation and Detection (SOLiD), they sequenced over 100 million small RNAs from human embryonic stem cell cultures and their neural precursor derivatives.
After feeding the sequence alignments into miRDeep, an algorithm that uses a probabilistic model of miRNA biogenesis to rank putative miRNAs based on position and frequency of the sequenced transcripts. The team went on to take a more functional look at the set by pulling down Ago2 complexes and see which of these predictions were actually slaving away in the RISC complex.
After all the dust settled, the team had identified:
- 800+ novel miRNA predictions
- 146 of these miRNA predictions that were associated Ago2 IP
- The 146 sequences associated with Ago2 showed MORE biologically-specific and LESS conserved expression profiles than most miRNAs identified to date
New Breed of Sequencing, A New Breed of miRNAs
Of equal cool factor as the novel sequences the team found, is their origin and behavior. The Rutgers crew looked into early stage development populations where, unlike in the more stable adult tissues, miRNAs can appear and disappear quicker than Yetti. Previous studies have uncovered handfuls of novel sequences in hESC, but nothing like this.Ron Hart, PI of the publishing lab at Rutgers, pointed to the sensitivity and accuracy of the SOLiD platform compared to the earlier deep sequencing methods used as a key factor. “The number of sequences we get back is a huge advantage, plus the two nucleotide color space interrogation combined with the colorspace alignment give us higher confidence with these types of smaller RNA sequences.”
Sequence conservation has been an accepted criteria for identifying novel miRNA for years now, but more research teams are producing evidence challenging the weight that should be assigned to sequence conservation. This latest data out of Rutgers definitely supports the case for using conservation cautiously, “We saw what appeared to be many rapidly evolving miRNAs. Most of these were conserved across primates, but not mammals,” explained Hart. “We can’t say more recently evolved sequences aren’t legitimate miRNA.”
Rock on Rutgers. Keep producing hits like this and you’ll give state icon The Boss, a run for his money. Check out all the data for yourself in PLoS One September 28th, 2009
And for all the details and technical specs for SOLiD™ Small RNA Analysis, check out ABI’s product page where they have tons of applications data.