Pippin Enriches for MicroRNAs in Zebrafish Transcriptome Study
Scientists from the Mayo Clinic, Institute for Systems Biology, and University of Illinois recently published a study investigating the effects of vitamin D on microRNA regulation of gene expression in zebrafish. The team used our Pippin Prep together with the NEBNext Multiplex Small RNA Kit from New England Biolabs to enrich their samples for miRNAs.
Lead authors Theodore Craig and Yuji Zhang performed RNA-seq with Illumina to look across the zebrafish transcriptome at which genes’ activity are changed by vitamin D. They found that by the seventh day of development, the zebrafish larvae have 2,500 protein-coding genes that are up- or down-regulated by vitamin D. The authors looked closely at the miRNAs involved in this process, finding that both known and novel miRNAs play a role. “The differentially expressed miRNAs are predicted to potentially alter mRNAs for metabolic enzymes, transcription factors, growth factors, and Jak-STAT signaling,” the authors write.
The scientists hypothesize that the higher number of miRNAs involved by day 7 of development is probably “due to the increased complexity of organs and the need for fine tuning of mRNA expression.”
You can view the paper, “Detection of 1,25-Dihydroxyvitamin D-Regulated miRNAs in Zebrafish by Whole Transcriptome Sequencing,” in the journal Zebrafish.
To learn more about the use of Pippin size selection to enrich miRNA selection with the NEBNext Multiplex Small RNA Kit, check out this app note.
Genome Editing Shows Potential for Shrinking Repeat Expansions
Several diseases, many of them involving neurological symptoms, have been linked to expansions of trinucleotide repeat sequences. These repeat expansion disorders include Fragile X syndrome, Huntington’s disease, and various forms of epilepsy and ataxia. In Fragile X, for example, a CGG pattern in the FMR1 gene is associated with the disease: people with 45 CGG copies or fewer are unaffected, while people with more than 200 copies have the syndrome.
This was the foundation for a fascinating project by researchers in France who used a transcription activator-like effector nuclease (TALEN) to target a CAG/CTG repeat in yeast cells, introducing a double-strand break at that location. By doing so, the repeat was deleted; multiple rounds of the TALEN genome editing activity led to repeat tracts with far fewer copies of the trinucleotides. Had these been human cells, the reduction in repeat size would have been enough to shorten a pathogenic region into a harmless region.
“Highly Specific Contractions of a Single CAG/CTG Trinucleotide Repeat by TALEN in Yeast,” published in PLoS One (Guy-Franck Richard et al.), details the promising results of using a TALEN for this type of work. It offers the first evidence that a TALEN can shrink a repeat expansion region, which could prove quite promising for biomedical researchers aiming to help people with repeat expansion disorders.
We were glad to see that the scientists used Pippin Prep for their size selection prior to sequencing on the Illumina platform. It’s an honor to be part of such a great effort!
Happy DNA Day!
Here at Sage Science headquarters, we’ll be spending April 25th celebrating DNA Day. If you’re not familiar, the day honors double milestones of the original publication of the structure of DNA in 1953 as well as the ceremonial completion of the Human Genome Project fifty years later.
Of course, thinking about DNA is nothing new for our team. After all, it takes a special kind of person to join a company like Sage, where success is measured by how well we can practice the art of sample prep. From our scientists to our sales representatives, we are constantly sharing ideas and feedback on ways to ensure that the highest-quality DNA is moved from sample to sequencer.
We’d love to take our technology back in time to Francis Crick and the rest of the lab in 1953 and see that extraordinary team react to what we take for granted today: completely automated, highly reproducible tools for selecting the exact DNA needed for study. Of course, their reliance on what we consider rudimentary tools to determine the structure of DNA makes the finding even more remarkable.
This year, perhaps DNA Day is a good time to consider what’s most needed in the life sciences field. Perhaps it’s sample prep standards as we move toward routine use of genomics in the clinic, or maybe it’s better legislation to improve on the nondiscrimination efforts of GINA. Feel free to share your own ideas below, and enjoy DNA Day
In the Journals: Pippin Publications
We’re pleased to see that the steady stream of publications citing the use of Pippin Prep and BluePippin as our automated DNA size selection tools help more and more users get more accurate and reproducible results. The instruments have been cited in some truly impressive publications, so our blog team took a tour of the literature to put together this roundup of recent activity. Enjoy!
SASI-Seq: sample assurance spike-ins, and highly differentiating 384 barcoding for Illumina sequencing
Michael Quail et al., BMC Genomics, 15:110
In this publication, scientists from the Wellcome Trust Sanger Institute and Frederick National Laboratory for Cancer Research describe Sample Assurance Spike-In sequencing, or SASI-Seq. With this method, scientists add uniquely barcoded amplicons to samples prior to library prep and sequencing; these barcodes allow them to deconvolute samples that get mixed up or to spot cross-contamination issues. The team used Pippin Prep to test barcode robustness through the size selection process, finding that barcodes were not affected.
Semiconductor-based DNA sequencing of histone modification states
Christine Cheng et al., Nature Communications 4:2672
Broad Institute scientists, along with collaborators at other institutions, provide optimized sample preparation protocols for generating ChIP-seq libraries on the Ion Torrent PGM. They show that Pippin size selection was required to generate usable libraries, even down to sub-nanogram input. Results were comparable to those from an Illumina ChIP-seq workflow.
Epigenetic Regulation of the DLK1-MEG3 MicroRNA Cluster in Human Type 2 Diabetic Islets
Vasumathi Kameswaran et al., Cell Metabolism (2014)
In this publication, researchers from the University of Pennsylvania and the Children’s Hospital of Philadelphia compared small RNAs from tissue samples gathered from people with and without type 2 diabetes. The scientists found evidence that a specific cluster of microRNAs was downregulated in diabetes patients, with the promoter of that locus hypermethylated. Pippin Prep was used to size-select microRNAs for sequencing.
Variant calling in low-coverage whole genome sequencing of a Native American population sample
Chris Bizon et al., BMC Genomics 2014, 15:85
Researchers from the University of North Carolina at Chapel Hill tested Thunder, a linkage disequilibrium-aware variant caller, on a community sample of Native Americans and determined that low-coverage whole genome sequencing is better at finding novel variants and associations than fixed-content genotyping arrays. They used Pippin Prep to select fragments of 300 base pairs for sequencing on the HiSeq 2000.
The Champion of Ancient Genomics: Sample Prep
A recent article in Nature News delves into the trends behind the rise of ancient genomics and finds that improvements in sample preparation have been essential to scientists’ success in this area.
As a team that focuses every day on new ways to make sample prep more robust, it was gratifying to see that advances in this section of the genomics workflow are making a real difference in what scientists can accomplish.
The article, “Human evolution: The Neanderthal in the family” from Ewen Callaway, describes the big challenge facing researchers looking to sequence genomes using DNA from fossils: getting enough sample that’s not too degraded to use. A decade ago, this was a major stumbling block. Today, the community has witnessed several papers describing genomic or mitochondrial sequences from a number of hominin fossils, mammoth and mastodon, and recently a horse that may have lived 700,000 years ago.
“Enabling this rush are technological improvements in isolating, sequencing and interpreting the time-ravaged DNA strands in ancient remains such as bones, teeth and hair,” Callaway writes. “Pioneers are obtaining DNA from ever older and more degraded remains, and gleaning insight about long-dead humans and other creatures.”
In the ancient horse project, for instance, scientists found new ways to improve DNA yield from the fossil. By lowering extraction temperature and making other small changes, the team boosted recovery 10-fold.
These sample prep advances are not only making it possible to sequence DNA from ancient remains, but they are also enabling virtually any lab to perform this kind of work, the article says. “New procedures mean that researchers can now reliably obtain DNA from all but the most degraded samples, and then sequence only the portions of a genome that they are interested in,” Callaway reports.
This is exactly the kind of impact that companies like ours hope to have in the genomics field — streamlining and improving the sample prep process to the point that scientists are no longer limited by these protocols. Congratulations to all the researchers and organizations who contributed to these tremendous advances for ancient genome studies!