Rapid microfluidic analysis of a Y-STR multiplex for screening of forensic samples.

In this paper, we demonstrate a rapid analysis procedure for use with a small set of rapidly mutating Y chromosomal short tandem repeat (Y-STR) loci that combines both rapid polymerase chain reaction (PCR) and microfluidic separation elements. The procedure involves a high-speed polymerase and a rapid cycling protocol to permit PCR amplification in 16 min. The resultant amplified sample is next analysed using a short 1.8-cm microfluidic electrophoresis system that permits a four-locus Y-STR genotype to be produced in 80 s. The entire procedure takes less than 25 min from sample collection to result. This paper describes the rapid amplification protocol as well as studies of the reproducibility and sensitivity of the procedure and its optimisation. The amplification process utilises a small high-speed thermocycler, microfluidic device and compact laptop, making it portable and potentially useful for rapid, inexpensive on-site genotyping. The four loci used for the multiplex were selected due to their rapid mutation rates and should proved useful in preliminary screening of samples and suspects. Overall, this technique provides a method for rapid sample screening of suspect and crime scene samples in forensic casework. Graphical abstract ᅟ.

Ultrafast STR Separations on Short-Channel Microfluidic Systems for Forensic Screening and Genotyping.

There are situations in which it is important to quickly and positively identify an individual. Examples include suspects detained in the neighborhood of a bombing or terrorist incident, individuals detained attempting to enter or leave the country, and victims of mass disasters. Systems utilized for these purposes must be fast, portable, and easy to maintain. DNA typing methods provide the best biometric information yielding identity, kinship, and geographical origin, but they are not portable and rapid. This study details the development of a portable short-channel microfluidic device based on a modified Agilent 2100 bioanalyzer for applications in forensic genomics. The system utilizes a denaturing polymer matrix with dual-channel laser-induced fluorescence and is capable of producing a genotype in 80 sec. The device was tested for precision and resolution using an allelic ladder created from 6 short tandem repeat (STR) loci and a sex marker (amelogenin). The results demonstrated a precision of 0.09-0.21 bp over the entire size range and resolution values from 2.5 to 4.1 bp. Overall, the results demonstrate the chip provides a portable, rapid, and precise method for screening amplified short tandem repeats and human identification screening.

High-throughput profiling of the serum N-glycome on capillary electrophoresis microfluidics systems: toward clinical implementation of GlycoHepatoTest.

We developed a 3 h procedure for preparing serum N-glycans and labeling them with 8-aminopyrene-1,3,6-trisulfonic acid (APTS) by sequential addition of reagents to the serum and incubation in a polymerase chain reaction (PCR) thermocycler. Moreover, we succeeded in analyzing these samples by capillary electrophoresis on three commercial microfluidics-based platforms: the MCE-202 MultiNA, the 2100 Bioanalyzer, and a modified prototype of the eGene system which were originally designed for nucleic acid separation and detection. Although these instruments use short separation channels, our technical improvements made it possible to reliably measure the N-glycans constituting GlycoHepatoTest. This test comprises a panel of biomarkers that allows follow-up of liver fibrosis patients starting from the early stage. In this way and for the first time, we demonstrate a clinical glycomics assay on an affordable, robust platform so that clinical chemistry laboratories can exploit glycomics in the diagnosis and monitoring of chronic liver disease. Another potential application is the rapid screening of the N-glycosylation of recombinant glycoproteins produced for pharmaceutical use.

The development of mini pentameric STR loci for rapid analysis of forensic DNA samples on a microfluidic system.

There is increasing interest in developing methods for portable DNA analysis in mass disasters and criminal identification. Currently most forensic STR DNA analysis is performed by CE; however, these instruments are not portable and require long sample run times. One potential solution is the development of microfluidic systems for DNA typing. Unfortunately, fairly long (ca. 20 cm) separation channels are usually required for the proper resolution of multiplexed STR loci used in human identification. Commercially available systems like the Agilent 2100 Bioanalyzer have a small footprint and utilize chips with shorter channels and reduced resolution. Such portable systems might be valuable for evidence screening in remote locations. However, due to their lower resolution, most standard 4 base STR loci and their inherent 2 base variants will not resolve on such systems. In this paper, we discuss the development of reduced length pentameric (5 base) STR amplicons. Pentameric STRs have fewer variant alleles and are easier to separate due to the wider spacing between alleles. By incorporating novel denaturing sieving polymers in a short microfluidic channel, we demonstrate efficient separations on these chips. Such an approach can serve as a useful tool for rapid microfluidic DNA typing.

Designing polymer matrix for microchip-based double-stranded DNA capillary electrophoresis.

Polyacrylamide (PAM) was used as a model polymer to build up an empirical model that relates polymer molecular weight, polymer concentration and solution viscosity. The desired random copolymers of acrylamide (AM) and N,N-dimethylacrylamide (DMA) used as DNA separation media for different specifications were synthesized under the guidance of the empirical model. The separation performances of rationally designed copolymers were tested in a 1.2 cm long separation channel, simulating microchip-based capillary electrophoresis. pBR322/HaeIII digest was successfully separated with good separation resolution and fast speed. Validation of the sieving ability of our polymers was performed in the Agilent 2,100 Bioanalyzer. The results of the 10 bp (base pair) DNA ladder separation demonstrate the potential of our approach for the sieving matrix in microchip-based electrophoresis.

Reversible thermo-responsive sieving matrix for oligonucleotide separation.

A reversible thermo-responsive gel system, consisting of Pluronic copolymer mixture of F87 and F127, has been used to successfully carry out the separation of oligonucleotides, for the first time, by microchip-based capillary electrophoresis. Pluronic triblock copolymers F87 (E(61)P(40)E(61)) and F127 (E(99)P(69)E(99)), with E, P, and subscript denoting oxyethylene, oxypropylene, and segment length respectively, have a unique temperature dependent viscosity-adjustable property and a dynamic coating ability in aqueous solution, including 1 x TBE buffer. The mixture solution has a reversible thermo-responsive property and its sol-gel transition temperature can be adjusted ranging from about 17 degrees C to 38 degrees C by varying the relative weight ratio of F87 and F127 at an optimized concentration of approximately 30% (w/v) for oligonucleotide separations. Oligonucleotide sizing markers ranging from 8 to 32 base could be successfully separated in a 1.5 cm long separation channel by the mixture solution in its gel-like state. A 30% (w/v) with a F87/F127 weight ratio of 1 ratio 2 which has a "sol-gel" transition point of about 26 degrees C shows the best sieving ability. The sieving ability of the mixture solution was further confirmed in an Agilent Bioanalyzer 2100 system. Fast separation of oligonucleotides has been achieved within 40 s with one base resolution.

The RIN: an RNA integrity number for assigning integrity values to RNA measurements.

BACKGROUND: The integrity of RNA molecules is of paramount importance for experiments that try to reflect the snapshot of gene expression at the moment of RNA extraction. Until recently, there has been no reliable standard for estimating the integrity of RNA samples and the ratio of 28S:18S ribosomal RNA, the common measure for this purpose, has been shown to be inconsistent. The advent of microcapillary electrophoretic RNA separation provides the basis for an automated high-throughput approach, in order to estimate the integrity of RNA samples in an unambiguous way. METHODS: A method is introduced that automatically selects features from signal measurements and constructs regression models based on a Bayesian learning technique. Feature spaces of different dimensionality are compared in the Bayesian framework, which allows selecting a final feature combination corresponding to models with high posterior probability. RESULTS: This approach is applied to a large collection of electrophoretic RNA measurements recorded with an Agilent 2100 bioanalyzer to extract an algorithm that describes RNA integrity. The resulting algorithm is a user-independent, automated and reliable procedure for standardization of RNA quality control that allows the calculation of an RNA integrity number (RIN). CONCLUSION: Our results show the importance of taking characteristics of several regions of the recorded electropherogram into account in order to get a robust and reliable prediction of RNA integrity, especially if compared to traditional methods.

Nrf2 transcription factor, a novel target of keratinocyte growth factor action which regulates gene expression and inflammation in the healing skin wound.

Keratinocyte growth factor (KGF) is a potent mitogen for epithelial cells, and it promotes survival of these cells under stress conditions. In a search for KGF-regulated genes in keratinocytes, we identified the gene encoding the transcription factor NF-E2-related factor 2 (Nrf2). Nrf2 is a key player in the cellular stress response. This might be of particular importance during wound healing, where large amounts of reactive oxygen species are produced as a defense against invading bacteria. Therefore, we studied the wound repair process in Nrf2 knockout mice. Interestingly, the expression of various key players involved in wound healing was significantly reduced in early wounds of the Nrf2 knockout animals, and the late phase of repair was characterized by prolonged inflammation. However, these differences in gene expression were not reflected by obvious histological abnormalities. The normal healing rate appears to be at least partially due to an up-regulation of the related transcription factor Nrf3, which was also identified as a target of KGF and which was coexpressed with Nrf2 in the healing skin wound. Taken together, our results reveal novel roles of the KGF-regulated transcription factors Nrf2 and possibly Nrf3 in the control of gene expression and inflammation during cutaneous wound repair.