Column-free, Caustic Solvent-free DNA or RNA Purification

Epicentre® (an Illumina Company) has developed a series of MasterPure™ DNA and RNA purification kits for nearly any sample type. While Epicentre’s QuickExtract™ DNA Extraction Solution is designed for the fastest extraction of low abundance DNA for PCR, the MasterPure™ kits yield the highest quality DNA and RNA for applications such as:

  • NGS genomic DNA and RNA-Seq library preps
  • DNA methylation/epigenomics studies
  • Genomic DNA and cDNA cloning
  • qPCR and qRT-PCR

These kits offer a simple protocol for the purification of Genomic DNA, cellular RNA, or total nucleic acid (TNA):

DNA RNA or Both

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rRNA depletion, poly(A) enrichment, or exonuclease treatment?

RNASeq studies are hampered by the pervasive excess of reads mapping to ribosomal RNA (rRNA), notorious for greatly reducing the amount of useful mRNA sequencing data. Here we highlight the advantages and disadvantages of the various approaches have been developed to address this problem.

Source: DOI: 10.1371/journal.pone.0096094

Source: DOI: 10.1371/journal.pone.0096094

 

1) rRNA depletion with magnetic beads: Technologies such as Ribo-zero™, Ribominus™, and MICROBExpress™ seem to be based on similar technology that uses rRNA depletion probes in combination with magnetic beads to deplete rRNAs from a sample, thus achieving the goal of enriching the representation of mRNAs. Based on our interactions with end-users, Ribo-zero™ customers have been quite pleased with this technology, although some have found it a bit pricey. As a result, some scientists have adapted home-made protocols either using the Organism-Specific Probe Selection software or by designing probes empirically based on the reads they see in their samples. In both cases users custom order Biotin-TEG DNA oligonucleotides in combination with BioMag® Nuclease-Free Streptavidin Particles to complete their own protocols.

2) Poly(A) enrichment: Scientists working in eurkaryotes find that simply purifying poly(A) mRNA with BioMag® Oligo (dT)20 Particles is the most cost-effective approach. The BioMag® SelectaPure mRNA System is a complete low cost kit for isolating mRNA. Clearly users wishing to study histone mRNAs or those of prokaryotes will have to find another solution.

3) rRNA depletion by nuclease: Some of the most straightforward approaches for rRNA depletion involve simple digestion of rRNA with Terminator™ 5′-Phosphate Dependent Exonuclease. While a mature mRNA will carry an m7GTP cap on its 5′ end protecting it from the 5′-to-3′ exonuclease activity, rRNAs and tRNAs carrying only a 5′ phosphate will be degraded. A number of published reports have used this exonuclease approach which seems to be particularly interesting when users have complex environmental samples with multiple prokaryotic species. Alternative strategies involve the use of DNA probes complementary to the rRNA prior to digestion with RNAse H, which degrades only the RNA strand in an RNA:DNA hybrid. RNase-Free DNase I is then used to eliminate the DNA Probes. A report in Nature Methods (DOI:10.1038/nmeth.2483) comparing multiple approaches for RNASeq library prep determined that the Hybridase™ Thermostable RNase H-based approach was superior for low quality, degraded RNA samples.

4) aRNA amplification: Very common in microarray protocols such as those using Illumina BeadChip, the linear amplification of antisense RNA (aRNA) also called cRNA seems like an interesting approach. Kits like the TargetAmp™TargetAmp™-Nano, and TargetAmp™-Pico aRNA Amplification Kits are designed to amplify the input poly(A) mRNA from very low input (down to 10pg) RNA samples such as those obtained by laser capture microdissected tissue or by flow sorting. After amplification, sufficient sample is present for microarray analyses of gene expression. Based on technology developed by James Eberwine for single cell analysis, the TargetAmp kits have been used in RNASeq protocols with very low quantity samples (For example see: Matsumura et al. and Li et al.).

TargetAmp

5) Rolling Circle Amplification: Similar to the Eberwine-based methods, Rolling Circle amplification protocols involve first the reverse transcription of mRNA with the goal of amplifying the cDNA, primarily for single-cell applications (see Pan et al.). These protocols are quickly gaining popularity and make use of CircLigase to circularize full-length single-stranded cDNA or T4 DNA Ligase kits such as Fast-Link™ DNA Ligation Kits for double-stranded cDNA circularization. Use of Phi29 DNA polymerase is then possible, despite the fact that Phi29 normally requires templates of at least 3-4kb. While it is clear to see how reverse transcription using oligo(dT) will enrich mRNA reads relative to those mapping to tRNA and rRNA, such an enrichment would not be possible in prokaryotes. The rolling circle amplification protocol has been adapted to transcriptome analysis in single prokaryotic cells by making use of random hexamers combined with Terminator™ 5′-Phosphate Dependent Exonuclease described in #3 above (see Kang et al.). Interestingly, the protocol makes use of thiophosphate-linked RNA random hexamers to reduce the formation of primer-dimers and to protect against Phi29 DNA polymerase exonuclease activity.

Diagram illustrating rolling circle DNA replication (credit: credit Madeleine Price Ball)

Diagram illustrating rolling circle DNA replication (credit: Madeleine Price Ball)

Need help building your protocol for enrichment of mRNA reads? Contact the technical specialists at tebu-bio!

Legal notes: TargetAmp™ and Ribo-zero™ are trademarks of Epicentre-Illumina. Ribominus™ and MicrobeExpress™ are trademarks of Life technologies.

High quality, low cost DNASeq kits and gene enrichment panels

Otogenetics' kits at tebu-bioOtogenetics Corporation, a DNASeq and RNASeq next generation sequencing service provider has processed over 11,000 samples and is now producing their own DNASeq kits. In order to reduce costs and improve results, Otogenetics developed and validated kits in-house and has now made these high quality, low cost kits available to other researchers.

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tebu-bio now distributes Epicentre-Illumina throughout Europe

tebu-bio’s 9 local offices throughout Europe have been bringing innovations to life science researchers for decades. For the past 8+ years, the company’s headquarters in France has served as the exclusive French distributor for Epicentre® Biotechnologies, a US-based company well-known for its unique enzymes and technologies for RNA biology work and Next Generation Sequencing Library prep. As of January 1, 2015, tebu-bio will be distributing Epicentre® products throughout Europe*.

tebu-bio is actively present throughout Europeepicentre_biotechnologies-tebu-bio-20120808162728

Among the most innovative Epicentre® products are those used for so called “tagmentation”, a process that allows researchers to produce libraries for DNASeq and RNASeq much more quickly. Indeed, Illumina®’s acquistion of Epicentre® and the company’s Nextera® DNASeq library prep kits was undoubtedly motivated by this recognized advantage. Similarly Epicentre®’s ScriptSeq Kits have reduced the time required to prepare RNASeq libraries from days to hours using a patented terminal tagging process. While kits and Ribo-Zero™ brand rRNA removal kits are extremely popular with sequencing platforms, RNA biologists and scientists wishing to customize their library prep rely heavily on Epicentre for their innovative enzymes.

For example, groups have published how one can use Epicentre®’s Ez-Tn5™ transposase enzyme to produce high quality DNASeq libraries. (see: doi:10.1101/gr.177881.114). Similarly, this enzyme is useful for tagmentation-based whole genome bisulfite sequencing for epigenetic studies, particularly when very little DNA is available (see: doi:10.1038/nprot.2013.118 and doi:10.1101/gr.136242.111).

Epicentre®’s CircLigase™ II is another favorite of researchers as it is able to attach the 5′ end of a single-stranded DNA molecule to it’s own 3′ end to allow so called rolling-circle replication and transcription for protocols such as iCLIP. Similarly, Epicentre®’s End-It™ DNA Repair and Fast-Link™ DNA Ligation Kits are useful for molecular biology work and library prep as they allow for the blunting and ligation of noncompatible or damaged double stranded DNA overhangs.

Epicentre®’s recent discontinuation of tobacco acid pyrophosphatase (TAP) has served as a reminder of how this company has routinely brought innovative enzymes to the RNA biology community. Among the hidden gems not yet discovered by many researchers are Epicentre®’s MasterPure™ DNA and RNA Purification Kits which function similar to TRIzol® in that they purify DNA or RNA without columns and yet they do not require dangerous organic solvents. Epicentre®’s MonsterScript™ RT Kits that compete with Superscript® III, are also worth trying.

tebu-bio’s distribution of the Epicentre® brand throughout Europe will create some unique bundling opportunities for European customers. Rather than purchasing a single enzyme from each supplier and paying for dry ice shipping multiple times, customers will be able to combine purchases from tebu-bio’s list of hundreds of brands. In particular, many of tebu-bio’s existing Epicentre® customers request combined quotes for enzymes and high quality custom modified oligonucleotides produced by TriLink™ Biotechnologies.

*Update May 6, 2015: tebu-bio’s distribution agreement of Epicentre® (an Illumina company) products includes Austria, Belgium, Bulgaria, Czech Republic, Denmark, Estonia, Finland, France, Germany, Greece, Hungary, Iceland, Italy, Latvia, Liechtenstein, Lithuania, Luxembourg, Malta, Monaco, Netherlands, Norway, Republic of Ireland, Republic of Cyprus, Romania, Poland Portugal, Slovakia, Slovenia, Spain, Sweden, Switzerland, and the United Kingdom.

Prevent adapter dimer formation during NGS library prep

from: Shore et al. Improved Small RNA Library Preparation Workflows for Next Generation Sequencing [Poster]

Sources: Shore et al. “Improved Small RNA Library Preparation Workflows for Next Generation Sequencing” [Poster]

It is now quite easy to prevent adapter dimer formation during NGS library preps by using chemically-modified adapters.

In a poster presented at the recent ASHG 2014 meeting in San Diego, researchers from TriLink Biotechnologies described their innovative technology solving the adapter dimer problem in Next Generation Sequencing library prep.

Similar to the primer dimer problem observed when performing PCR (i.e. low molecular weight PCR by-products of primer-primer hybridization), adapter dimerization generates by products of NGS library prep that will greatly decrease the number of informative sequencing reads. This is particularly a problem for low-input studies and when working with small RNAs (e.g. miRNA).

TriLink’s expertise in chemistry-based solutions for biological problems has previously led to the creation of the CleanAmp™ product line. CleanAmp™ PrimersCleanAmp™ dNTPs and 2X PCR Master Mix employ heat labile modifications, similar to those on hot-start polymerases, to prevent primer dimerization or premature polymerase activity prior to a high-temperature activation step.

CleanAmp™ 7-deaza-dGTP is a chemistry-based solution allowing for the amplification of difficult GC-rich PCR products.

Interested in learning more about TriLink’s products and services (including GMP production of modified oligonucleotides for Phase I human trials)? Contact tebu-bio experts who are representing TriLink in Europe.

Organism-Specific Probe Selection and rRNA depletion

As detailed in a previous post, rRNA depletion with kits such as Ribo-zero greatly increases the number of usable reads in RNASeq studies, but this one-size-fits-all approach is not appropriate for some researchers. In eurkaryotic studies, researchers often find that poly(A) enrichment using the BioMag® SelectaPure mRNA System making use of BioMag® Oligo (dT)20 Particles is a cost-effective means to separate mRNA from rRNA and tRNA. Prokaryotic mRNAs are not polyadenylated however, so poly(A) enrichment is not a possibility for researchers working with microorganisms. Although there exist rRNA depletion kits for bacteria, the organism-to-organism variability in rRNA sequences may lead to suboptimal depletion in some species and even the unintended depletion of specific mRNAs.

An article published in PLoS ONE (DOI: 10.1371/journal.pone.0074286) has described a computer program specifically for the purpose of designing rRNA depletion probes for various organisms. Using the example of the 16S and 23S rRNAs of Mycobacterium smegmatis, the authors report improved mRNA integrity and abundance using this approach compared to using MICROBExpress™.

The organism-specific probe selection software is available for download here. Once designed, researchers can purchase Biotin-TEG DNA oligonucleotides and BioMag® Nuclease-Free Streptavidin Particles for efficient depletion of rRNA from their organism of choice.

Organism-Specific Probe Selection and rRNA depletion. doi:10.1371/journal.pone.0074286.g001

Organism-Specific Probe Selection and rRNA depletion.
doi:10.1371/journal.pone.0074286.g001