Diagnostic plots for detecting outlying slides in a cDNA microarray experiment.

Park, Taesung,Yi, Sung-Gon,Lee, SeungYeoun,Lee, Jae K Eaton Pub. Co 2005 Biotechniques Vol.38 No.3

<P>Different sources of systematic and random error variations are often observed in cDNA microarray experiments. A simple scatter plot is commonly used to examine outlying slides that have unusual expression patterns or larger variability than other slides. These outlying slides tend to have large impacts on the subsequent analyses, such as identification of differentially expressed genes and clustering analysis. However, it is difficult to select outlying slides rigorously and consistently based on subjective human pattern recognition on their scatter plots. A graphical method and a rigorous diagnostic measure are proposed to detect outlying slides. The proposed graphical method is easy to implement and shown to be quite effective in detecting outlying slides in real microarray data sets. This diagnostic measure is also informative to compare variability among slides. Two cDNA microarray data sets are carefully examined to illustrate the proposed approach. A 3840-gene microarray experiment for neuronal differentiation of cortical stem cells and a 2076-gene microarray experiment for anticancer compound time-course expression of the NCI-60 cancer cell lines.</P>

A convenient and efficient purification method for chemically labeled oligonucleotides.

Hwang, Jihee,Kang, Junhee,Kim, Seong Keun,Kim, Younggyu Eaton Pub. Co 2013 Biotechniques Vol.54 No.5

<P>We developed an efficient, cost-effective, and rapid purification method for chemically-labeled oligonucleotides that requires less time than conventional procedures such as ethanol precipitation or size-exclusion chromatography. Based on the hydrophilic and hydrophobic properties of DNA and amine-reactive fluorophores, we show that n-butanol saturated with distilled water may be used to remove unreacted fluorophores by sequestering them in the organic phase, while labeled DNA remains in the aqueous phase. This phase extraction method is simple, fast, and allows for processing multiple samples simultaneously, a necessity for high-throughput labeling strategies.</P>

Transposon-directed base-exchange mutagenesis (TDEM): a novel method for multiple-nucleotide substitutions within a target gene.

Kim, Yun Cheol,Lee, Hui Sun,Yoon, Sukjoon,Morrison, Sherie L Eaton Pub. Co 2009 Biotechniques Vol.46 No.7

<P>In this report we describe transposon-directed base-exchange mutagenesis (TDEM), an efficient and controllable method for introducing a mutation into a gene. Each round of TDEM can remove up to 11 base pairs from a randomly selected site within the target gene and replace them with any length of DNA of predetermined sequence. Therefore, the number of bases to be deleted and inserted can be independently regulated providing greater versatility than existing methods of transposon-based mutagenesis. Subsequently, multiple rounds of mutagenesis will provide a diverse mutant library that contains multiple mutations throughout the gene. Additionally, we developed a simple frame-checking procedure that eliminates nonfunctional mutants containing frameshifts or stop codons. As a proof of principle, we used TDEM to generate mutant lacZalpha lacking alpha-complementation activity and recovered active revertants using a second round of TDEM. Furthermore, a single round of TDEM yielded unique, inactive mutants of ccdB.</P>

Continuous-exchange cell-free protein synthesis using PCR-generated DNA and an RNase E-deficient extract.

Jun, Soo Youn,Kang, Sang Hyeon,Lee, Kwang-Ho Eaton Pub. Co 2008 Biotechniques Vol.44 No.3

<P>Though the use of PCR-generated DNA (i.e., linear template) as template DNA is desirable because of its simple preparation, the linear template has not been routinely used in a conventional continuous-exchange cell-free (CECF) protein synthesis system due to the instability of the linear template and/or its transcript in the relatively long operation period. To overcome this problem and enhance soluble protein yield, an RNase E-deficient and molecular chaperone-enriched extract was used: (i) for compensating for the decrease in messenger RNA (mRNA) levels transcribed from the unstable linear template with improvement of mRNA stability by depletion of RNase E activity; and (ii) for enhancement of the soluble protein portion by assisting of the molecular chaperones. As a result, soluble erythropoietin production from a linear template was significantly enhanced in this modified CECF system using the RNase E-deficient and molecular chaperone-enriched extract, and the amount of soluble erythropoietin was estimated to be roughly 70% of that from a circular plasmid. We can conclude that the use of RNase E-deficient and molecular chaperone-enriched S30 extract mixture is effective in the enhancement of soluble protein expression from a linear template in the CECF system.</P>

Purification of inclusion body-forming peptides and proteins in soluble form by fusion to Escherichia coli thermostable proteins.

Thapa, Arjun,Shahnawaz, Md,Karki, Pratap,Raj Dahal, Giri,Sharoar, Md Golam,Yub Shin, Song,Sup Lee, Jung,Cho, Byungyun,Park, Il-Seon Eaton Pub. Co 2008 Biotechniques Vol.44 No.6

<P>Proteins and peptides expressed in the prokaryotic system often form inclusion bodies. Solubilization and refolding procedures can be used for their recovery, but this process remains difficult. One strategy for improving the solubility of a protein of interest is to fuse it to a highly soluble protein. To select a suitable fusion partner capable of solubilizing the aggregation-prone (inclusion body-forming) proteins and peptides, Escherichia coli thermostable proteins were identified and tested. Among them, trigger factor (TF) protein was selected because of its high expression and stability. Using an expression system based on fusion to TF, selected proteins and peptides that otherwise form inclusion bodies were expressed in soluble state and were purified like other soluble proteins. This system provides a convenient method for production of aggregation-prone proteins and peptides.</P>

Three-dimensional migration of neutrophils through an electrospun nanofibrous membrane.

Jin, Songwan,Park, Tae-Min,Kim, Cho-Hee,Kim, Jin-Soo,Le, Binh Duong,Jeong, Young Hun,Kwak, Jong-Young,Yoon, Sik Eaton Pub. Co 2015 Biotechniques Vol.58 No.6

<P>The study of immune cell migration is important for understanding the immune system network, which is associated with the response to foreign cells. Neutrophils act against foreign cells before any other immune cell, and they must be able to change shape and squeeze through narrow spaces in the extracellular matrix (ECM) during migration to sites of infection. Conventional in vitro migration assays are typically performed on two-dimensional substrates that fail to reproduce the three-dimensional (3-D) nature of the ECM. Here we present an in vitro method to simulate the 3-D migration of neutrophils using an electrospun nanofibrous membrane, which is similar to the ECM in terms of morphology. We examined the properties of neutrophil movement and the effects of gravity and the presence of IL-8, which has been widely used as a chemotactic attractant for neutrophils. The number of neutrophils passing through the nanofibrous membrane were higher, and their movement was more active in the presence of IL-8. Also, we confirmed that neutrophils could migrate against gravity toward IL-8 through a nanofibrous membrane.</P>

Performance evaluation of thermal cyclers for PCR in a rapid cycling condition.

Kim, Young Ho,Yang, Inchul,Bae, Young-Seuk,Park, Sang-Ryoul Eaton Pub. Co 2008 Biotechniques Vol.44 No.4

<P>The performance of thermal cyclers for polymerase chain reactions (PCR) is of great concern in terms of the reliability of PCR-based assays, particularly when rapid cycling conditions are applied to small volume reactions. In this work, the precision of the temperature controls during rapid thermal cycling was measured in 19 commercial thermal cyclers of 8 different models. The temperatures of test solutions in specific locations in each thermal block were simultaneously monitored at 1 s intervals during thermal cycling. A temperature-sensitive multiplex PCR was run in parallel to assess undesirable PCR results caused by poor temperature control. Under the given conditions (20 s of annealing time and 20 microL reaction volume), a majority of the tested instruments showed prominent curving, undershooting, and/or overshooting in their temperature profiles, which substantially influenced the results of the temperature-sensitive multiplex PCR. Variations between wells were also observed in most instruments. It is strongly hoped that these problems will be addressed by manufacturers and that they will make substantial improvements in the precision and efficiency of thermal cyclers. In the meantime, users of thermal cyclers might be able to avoid unexpected poor outcomes of sensitive PCR-based assays by designing their PCR protocols with these findings in mind.</P>