For over 27 years, BioTechniques has published exceptional peer-reviewed research articles that describe innovative methods, modifications to existing methods, or innovative applications. But to sum up the audience favorites of last year alone, we’ve compiled the list of 2010’s most-viewed articles on BioTechniques.com.
We hope this list provides you with a chance to see which methods are of most interest to the broader life science research community, and which have had the largest impact on our audience.
As we enter 2011 with a fresh lineup of techniques and methods to publish, we hope you continue telling us your favorites–follow us on Twitter, connect with us on Facebook, subscribe to our e-mail newsletters, and share our articles with your colleagues.
1. Overlap extension PCR cloning: a simple and reliable way to create recombinant plasmids. June 2010
In our June 2010 issue, Bryksin and Matsumura from Emory University achieved a simple, efficient, and relatively foolproof method of cloning an insert of choice into a plasmid without the use of restriction endonucleases or T4 DNA ligase. The researchers created overlapping sequences within the vector using PCR amplification of a linear insert combined with chimeric primer sequences. In a second PCR reaction, the insert was effectively employed as a mega primer; the hybridized insert was extended by Phusion DNA polymerase using vector as a template until the 5′ end of the vector is reached.
2. Antibody validation. March 2010
When it comes to the bare necessities of basic science research and clinical
assays, antibodies are an absolute staple in any researchers’ reagent
arsenal. However, with such an abundance of commercially produced
antibodies available and the lack of universally accepted guidelines for
determining their validity, one inevitably asks the question, “Is antibody
there?” In this review, Yale University School of Medicine researchers
Bordeaux et al. highlighted common practices and procedures for validating
antibodies, along with methods for troubleshooting and avoiding common
pitfalls.
3. Interference with spectrophotometric analysis of nucleic acids and proteins by leaching of chemicals from plastic tubes. April 2010
Absorbance spectrophotometry is a routinely used procedure in the
quantification and quality assessment of nucleic acids and proteins within
solutions. However, Lewis et al. showed that the leaching of
light-absorbing chemicals from commonly used polypropylene tubes used to
store samples can interfere with spectrophotometric measurements.
4. Purifying natively folded proteins from inclusion bodies using sarkosyl, Triton X-100, and CHAPS. January 2010
A simple, fast, and effective solution to the interminable dilemma of
recovering fusion proteins from inclusion bodies was finally developed in
early 2010. Here, Tao et al. demonstrate for the first time that a
combination of three detergents—sarkosyl, Triton X-100, and
CHAPS—significantly improved binding efficiency of GST and GST-fusion
proteins to glutathione (GSH) Sepharose.
5. Direct PCR amplification and sequencing of specimens' DNA from preservative ethanol. March 2010
Making a clever, albeit debaucherous segue into discussing ethanol
preservation of tissue, Shokralla et al. were able to recover amplifiable
quantities of caterpillar DNA from mescal, the alcoholic beverage in which
it was stored. These results suggest that commonly used DNA extraction
methods such as tissue homogenization and sonication may prove redundant and
that preservative ethanol can be used as a source of genetic material for
noninvasive sampling when little or no tissue is available for genetic
analysis.
6. Improving sequencing quality from PCR products containing long mononucleotide repeats. April 2010
Despite the necessity for accurate determination of PCR product sequencing,
little has been done to mitigate the formation of stutter products, a common
artifact in the PCR amplification of frequently used genetic markers. These
stutters are generally the result of slipped-strand mispairing during
replication of mononucleotide repeats, short repetitive motifs composed of
eight or more nucleotides. In this report, Fazekas et al. tested the
effects of altering various PCR parameters on sequence quality for a set of
sequences containing mononucleotide A/T repeats of 10–17 bp.
7. High-throughput methods to define complex stem cell niches. April 2010
Unique among all other cell types is the capability of stem cells to
self-renew and produce differentiated progeny. These defining endowments
make them an extremely interesting study candidate for clinical and
pharmaceutical applications. However, due to a lack of in vitro models that
effectively mimic the in vivo stem cell environment, or niche, the potential
of stem cells in clinics and as a diagnostic tool has remained an untapped
resource. This dilemma has prompted intense development and application of
microfabrication and micropatterning technologies in stem cell biology. In
the special BioTechniques Focus: Cell Culture Technology section
published in our April 2010 issue, Kobel and Lutolf review these tools that
can ultimately be used to examine the complex molecular interplay of stem
cells and their niche.
8. Controlling transcription with noncoding RNAs in mammalian cells. June 2010
Emerging research suggests that long noncoding RNAs (ncRNA) may play a
critical role in the basic fabric of gene regulation in the human cell. In
a special review, Turner and Morris detail the current state of research
regarding gene regulation by ncRNAs, as well as current molecular techniques
and the potential for future therapeutic applications.
To freeze or not to freeze, that is the question . . . or more aptly, to freeze or paraffin-embed. In this study, Sánchez-Navarro et al. conducted a side by side comparison, quantifying relative RNA expression of genes from fresh frozen (FF) or formalin fixed paraffin embedded (FFPE) tumor tissue. Although total RNA extracted from FFPE samples presented a shift in raw cycle threshold (Cq)—representative of extensive RNA degradation—the results were more apparent than real. To wit, proper normalization compensated for the effects of RNA degradation in gene expression measurements.
10. Concordance among digital gene expression, microarrays, and qPCR when measuring differential expression of microRNAs. March 2010
Profiling microRNA (miRNA) expression has quickly advanced to the forefront of molecular research given their critical role in many cellular functions. Several methods are commonly used to achieve miRNA profiling: hybridization-based (microarrays), sequencing-based, or amplification-based (quantitative reverse transcription-PCR, qPCR). However, inherent in microarray technology is the inability to accurately distinguish between mature and immature miRNA forms. In response, vendors have developed different methods to resolve this issue. In this study, Pradervand et al. compared differential mRNA expression using the Affymetrix, Agilent, and Illumina microarray platforms as well as qPCR (Applied Biosystems) and ultra high–throughput sequencing (Illumina).
---
留言列表
