A rapid and low-cost platform for detection of bacterial based on microchamber PCR microfluidic chip.

Polymerase chain reaction (PCR) has been considered as the gold standard for detecting nucleic acids. The simple PCR system is of great significance for medical applications in remote areas, especially for the developing countries. Herein, we proposed a low-cost self-assembled platform for microchamber PCR. The working principle is rotating the chamber PCR microfluidic chip between two heaters with fixed temperature to solve the problem of low temperature variation rate. The system consists of two temperature controllers, a screw slide rail, a chamber array microfluidic chip and a self-built software. Such a system can be constructed at a cost of about US$60. The micro chamber PCR can be finished by rotating the microfluidic chip between two heaters with fixed temperature. Results demonstrated that the sensitivity of the temperature controller is 0.1℃. The relative error of the duration for the microfluidic chip was 0.02 s. Finally, we successfully finished amplification of the target gene of Porphyromonas gingivalis in the chamber PCR microfluidic chip within 35 min and on-site detection of its PCR products by fluorescence. The chip consisted of 3200 cylindrical chambers. The volume of reagent in each volume is as low as 0.628 nL. This work provides an effective method to reduce the amplification time required for micro chamber PCR.

Design and fabrication of portable continuous flow PCR microfluidic chip for DNA replication.

The reasons for restricting continuous flow polymerase chain reaction (CF-PCR) microfluidic chip from lab to application are that it is not portable and requires costly external precision pumps for sample injection. Herein, we employed water as the substitute for PCR solution, and investigated the effect of the cross-section, width-to-depth ratio, and the length ratio for three temperature zones of the micro channel on the thermal and flow distribution of fluid in micro tube by finite element analysis. Results show that the central velocity is uniform and stable velocity occupies the most if the cross-section is rectangular. The deviation between predefined temperature and theoretical temperature is slight and the fluid flux is the most if width-to-depth ratio is 1:1. It is suitable for the short DNA replication if the high temperature zone Wh is larger than the low temperature zone Wl, and vice versa. Then a portable CF-PCR microfluidic chip was fabricated and an automatic sample injection system was developed. As an application, we have successfully amplified the DNA of Treponema denticola in the chip within 8 min. Such a study may offer new insight into the design of CF-PCR microfluidic chip and promote it from lab-scale research to full-scale application.

Simultaneous Separation and Purification of Five Polymethoxylated Flavones from "Dahongpao" Tangerine (Citrus tangerina Tanaka) Using Macroporous Adsorptive Resins Combined with Prep-HPLC.

In this study, a preparative separation method was established to simultaneously isolate the polymethoxylated flavones (PMFs) from the peel of "Dahongpao" tangerine using macroporous adsorptive resins (MARs) combined with prep-HPLC. The total PMFs were enriched using MARs to remove most sugars, water-soluble pigments, and flavanones, and the eluents obtained were analyzed by ultra-performance liquid chromatography (UPLC) to determine the PMF composition. The separation and purification of PMFs were carried out by using a mass spectrometry-guided prep-HPLC with a gradient elution of acetonitrile-water (v/v), simultaneously. The purity of these PMFs was determined by UPLC, and their chemical structures were confirmed by electrospray ionization mass spectrometry (ESI-MS-MS), ultraviolet (UV), and nuclear magnetic resonance (NMR). Using the present method, five PMFs, including 5,6,7,4'-tetramethoxyflavone (1), nobiletin (2), tangeretin (3), sinensetin (4), and 5-hydroxy-6,7,8,3',4'-pentamethoxyflavone (5), can be purified simultaneously, and the purity of the compounds obtained were 95.3%, 99.7%, 99.5%, 98.9%, and 98.1%, respectively. The method reported here is simple, rapid, and efficient, and it can be used to separate PMFs from citrus fruit peels and, potentially, other plant materials.

Gene analysis of multiple oral bacteria by the polymerase chain reaction coupled with capillary polymer electrophoresis.

Capillary polymer electrophoresis is identified as a promising technology for the analysis of DNA from bacteria, virus and cell samples. In this paper, we propose an innovative capillary polymer electrophoresis protocol for the quantification of polymerase chain reaction products. The internal standard method was modified and applied to capillary polymer electrophoresis. The precision of our modified internal standard protocol was evaluated by measuring the relative standard deviation of intermediate capillary polymer electrophoresis experiments. Results showed that the relative standard deviation was reduced from 12.4-15.1 to 0.6-2.3%. Linear regression tests were also implemented to validate our protocol. The modified internal standard method showed good linearity and robust properties. Finally, the ease of our method was illustrated by analyzing a real clinical oral sample using a one-run capillary polymer electrophoresis experiment.

Analysis of small interfering RNA by capillary electrophoresis in hydroxyethylcellulose solutions.

The analysis of small interfering RNA (siRNA) is important for gene function studies and drug developments. We employed CE to study the separation of siRNA ladder marker, which were ten double-stranded RNA (dsRNA) fragments ranged from 20 to 1000 bp, in solutions of hydroxyethylcellulose (HEC) polymer with different concentrations and molecular weights (Mws). Migration mechanism of dsRNA during CE was studied by the mobility and resolution length (RL) plots. We found that the RL depended on not only the concentration of HEC, but also the Mw of HEC. For instance, RL of small dsRNA fragment was more influenced by concentration of high Mw HEC than large dsRNA fragment and RL of large dsRNA fragment was more influenced by concentration of low Mw HEC than small dsRNA fragment. In addition, we found electrophoretic evidence that the structure of dsRNA was more compact than dsDNA with the same length. In practice, we succeeded to separate the glyceraldehyde 3-phosphate dehydrogenase siRNA in the mixture of the siRNA ladder marker within 4 min.

Electrophoresis of periodontal pathogens in poly(ethyleneoxide) solutions with uncoated capillary.

Periodontitis is a prevalent inflammatory disease caused by different species of anaerobic bacteria such as Porphyromonas gingivalis (P.g), Treponema denticola (T.d), and Tannerella forsythia (T.f). We compared the separation result of DNA ladders in hydroxyethyl cellulose, poly(ethyleneoxide) (PEO), and polyethylene glycol and analyzed the effect of polymer concentration, electric field, and temperature of the background electrolyte on the separation performance. Results demonstrated that there was a linear relationship (R=0.942) for 100 to 700bp of DNA and its migration time. Finally, the polymerase chain reaction products of P.g, T.d, and T.f were successfully identified within 8.5 min in 0.5% PEO with uncoated capillary.

Simultaneous amplification of DNA in a multiplex circular array shaped continuous flow PCR microfluidic chip for on-site detection of bacterial.

Based on time to place conversion, continuous flow polymerase chain reaction (CF-PCR) can realize a rapid amplification of DNA by running the PCR reagent in a serpentine microchannel but a larger space is required for each sample, which greatly reduces the efficiency of the CF-PCR. Herein, we propose a multiplex circular array shaped CF-PCR microfluidic chip for on-site detection of bacteria. There were 12 serpentine microchannels which were distributed on the disc in an annular form, and each microchannel consisted of an inlet for sample injection, and an outlet for the detection of the PCR products based on fluorescence. Samples could be simultaneously driven into each inlet by a one-to-twelve diverter through a syringe. Moreover, the method of adding fluorescent dyes at the end of the microchannel can solve the inhibition effect of excessive fluorescent dyes on the PCR reaction. The process finished with simultaneous amplification of 12 different target genes from Porphyromonas gingivalis, Treponema denticola, Tannerella forsythia, and Escherichia coli, and on-site detection of their corresponding positives within 23 min. The fastest detectable PCR reaction time was 5.38 ± 0.2 min at a flow rate of 1 mL h-1. For E. coli, the minimum detectable concentration was 2.5 × 10-3 ng µL-1 in this microfluidic system. Such a system can increase the throughput of CF-PCR for point-of-care testing of pathogens.

Lower fluidic resistance of double-layer droplet continuous flow PCR microfluidic chip for rapid detection of bacteria.

BACKGROUND: Rapid diagnosis of harmful microorganisms demonstrated its great importance for social health. Continuous flow PCR (CF-PCR) can realize rapid amplification of target genes by placing the microfluidic chip on heaters with different temperature. However, bubbles and evaporation always arise from heating, which makes the amplification not stable. Water-in-oil droplets running in CF-PCR microfluidic chip with uniform height takes long time because of the high resistance induced by long meandering microchannel. To overcome those drawbacks, we proposed a double-layer droplet CF-PCR microfluidic chip to reduce the fluidic resistance, and meanwhile nanoliter droplets were generated to minimize the bubbles and evaporation. RESULTS: Experiments showed that (1) fluidic resistance could be reduced with the increase of the height of the serpentine microchannel if the height of the T-junction part was certain. (2) Running speed, the size and the number of generated droplets were positively correlated with the cross-sectional area of the T-junction and water pressure. (3) Droplet fusion happened at higher water pressure if other experimental conditions were the same. (4) 0.032 nL droplet was created if the cross-sectional area of T-junction and water pressure were 1600 µm2 (40 × 40 µm) and 7 kPa, respectively. Finally, we successfully amplified the target genes of Porphyromonas gingivalis within 11'16″ and observed the fluorescence from droplets. SIGNIFICANCE AND NOVELTY: Such a microfluidic chip can effectively reduce the high resistance induced by long meandering microchannel, and greatly save time required for droplets CF-PCR. It offers a new way for the rapid detection of bacterial.

Micropatterned thermoresponsive surfaces by polymerization of monomer crystals: modulating cellular morphology and cell-substrate interactions.

A novel and facile approach has been developed to create thermoresponsive surfaces with macroscale patterns together with microscale features. The surface patterns were formed by applying macroscale nucleation agent patterns onto saturated N-isopropylacrylamide monomer solution membranes to induce the divergent growth of needlelike monomer crystals; the patterned monomer crystals were then photopolymerized to form patterned thermoresponsive films. A series of analytical tools (i.e., scanning electron microscopy, profilometry, and contact angle measurement) were used to characterize the properties of the patterned films. Cell coculture on this patterned thermoresponsive films enables cell separation and sorting by modulating temperature- and topography-dependent cell-substrate interactions and cell morphology, respectively. This versatile technique allows the formation of various macroscale patterns with microscale features over large areas, and on most solid substrates, within minutes, all of this without the need for expensive equipment and facilities. Such patterned surfaces can act as both in vitro tumor models and separation platforms for cancer studies. This method can also be applied to other cell-based biological studies and clinical applications.

A thermosensitive hydrogel capable of releasing bFGF for enhanced differentiation of mesenchymal stem cell into cardiomyocyte-like cells under ischemic conditions.

A thermosensitive hydrogel capable of differentiating mesenchymal stem cells (MSCs) into cardiomyocyte-like cells was synthesized. The hydrogel was based on N-isopropylacrylamide (NIPAAm), N-acryloxysuccinimide, acrylic acid, and hydroxyethyl methacrylate-poly(trimethylene carbonate). The hydrogel was highly flexible at body temperature with breaking strain >1000% and Young's modulus 45 kPa. When MSCs were encapsulated in the hydrogel and cultured under normal culture conditions (10% FBS and 21% O(2)), the cells differentiated into cardiomyocyte-like cells. However, the differentiation was retarded, and even diminished, under low nutrient and low oxygen conditions, which are typical of the infarcted heart. We hypothesized that enhancing MSC survival under low nutrient and low oxygen conditions would restore the differentiation. To enhance cell survival, a pro-survival growth factor (bFGF) was loaded in the hydrogel. bFGF was able to sustainedly release from the hydrogel for 21 days. Under the low nutrient and low oxygen conditions (1% O(2) and 1% FBS), bFGF enhanced MSC survival and differentiation in the hydrogel. After 14 days of culture, survival of 70.5% of MSCs remained in the bFGF-loaded hydrogel, while only 4.9% of MSCs remained in the hydrogel without bFGF. The differentiation toward cardiomyocyte-like cells was completely inhibited at 1% FBS and 1% oxygen. Loading bFGF in the hydrogel restored the differentiation, as confirmed by the expression of cardiac markers at both the gene (MEF2C and CACNA1c) and protein (cTnI and connexin 43) levels. bFGF loading also up-regulated the paracrine effect of MSCs. VEGF expression was significantly increased in the bFGF-loaded hydrogel. These results demonstrate that the developed bFGF-loaded hydrogel may potentially be used to deliver MSCs into hearts for regeneration of heart tissue.

Creating 3D angiogenic growth factor gradients in fibrous constructs to guide fast angiogenesis.

Fast angiogenesis in 3D fibrous constructs that mimic the morphology of the extracellular matrix remains challenging due to limited porosity in the densely packed constructs. We investigated whether mimicking the in vivo chemotaxis microenvironment for native blood vessel formation would stimulate angiogenesis in the fibrous constructs. The chemotaxis microenvironment was created by introducing 3D angiogenic growth factor gradients into the constructs. We have developed a technique that can quickly fabricate (∼40 min) such 3D gradients by simultaneously electrospinning polycaprolactone (PCL) fibers, encapsulating gradient amount of bFGF (stabilized by heparin) into poly(lactide-co-glycolide) (PLGA) microspheres, and electrospraying the microspheres into PCL fibers. Gradient formation was confirmed by fluorescence microscopy. Gradients with different steepnesses were obtained by modulating the initial concentration of the bFGF solution. All of the constructs were able to sustainedly release bioactive bFGF over a 28 day period. The release kinetics was dependent on the bFGF loading and steepness of the gradient. In vitro cell migration study demonstrated that bFGF gradients significantly increased the depth of cell migration. To assess the efficacy of bFGF gradients in inducing angiogenesis, we implanted constructs subcutaneously using mouse model. bFGF gradients significantly promoted cell penetration into the constructs. After 10 days of implantation, a high density of mature blood vessels (positive to both CD31 and α-SMA) were formed in the constructs. Vessel density was increased with the increase in steepness of the bFGF gradient. These gradient constructs may have potential to engineer vascularized tissues for various applications.

A continuous flow PCR array microfluidic chip applied for simultaneous amplification of target genes of periodontal pathogens.

The concept of time to place conversion makes using a continuous flow polymerase chain reaction (CF-PCR) microfluidic chip an ideal way to reduce the time required for amplification of target genes; however, it also brings about low throughput amplicons. Although multiplex PCR can simultaneously amplify more than one target gene in the chip, it may easily induce false positives because of cross-reactions. To circumvent this problem, we herein fabricated a microfluidic system based on a CF-PCR array microfluidic chip. By dividing the chip into three parts, we successfully amplified target genes of Porphyromonas gingivalis (P.g), Tannerella forsythia (T.f) and Treponema denticola (T.d). The results demonstrated that the minimum amplification time required for P.g, T.d and T.f was 2'07'', 2'51'' and 5'32'', respectively. The target genes of P.g, T.d and T.f can be simultaneously amplified in less than 8'05''. Such a work may provide a clue to the development of a high throughput CF-PCR microfluidic system, which is crucial for point of care testing for simultaneous detection of various pathogens.

Capillary electrophoresis of DNA with high resolution based on copoly(pentaerythritoltetra succinimidylcarboxypentyl/aminopropyl polyoxyethylene) hydrogel.

Capillary gel electrophoresis is widely applied for determination of sequence and size of DNA, in which the sieving gel plays an unignorable role. Herein, a pore-size controllable hydrogel was synthesized in the capillary with two symmetrical tetrahedron-like macromonomers consisting of pentaerythritoltetra (succinimidylcarboxypentyl) polyoxyethylene (PS) and pentaerythritoltetra (aminopropyl) polyoxyethylene) (PA). By capillary electrophoresis of the DNA fragments with this hydrogel, it is found that a homogenous structure of hydrogel which is more suitable for the DNA separation can be achieved when the molecular weight of PA is approximate to that of PS. DNA fragments smaller than 1500 bp can be well resolved in this hydrogel within 13 min. More than 100 consecutive runs can be carried out in such a dynamically coated capillary before performance begins to degrade. Notably, such hydrogel can realize separation of dsDNA up to single base pair resolution and same length of dsDNA with 1 bp difference.

Multiplex amplification of target genes of periodontal pathogens in continuous flow PCR microfluidic chip.

Porphyromonas gingivalis (P.g), Treponema denticola (T.d), and Tannerella forsythia (T.f) are believed to be the major periodontal pathogens that cause gingivitis, which affects 50-90% of adults worldwide. Microfluidic chips based on continuous flow PCR (CF-PCR) are an ideal alternative to a traditional thermal cycler, because it can effectively reduce the time needed for temperature transformation. Herein, we explored multi-PCR of P.g, T.d and T.f using a CF-PCR microfluidic chip for the first time. Through a series of experiments, we obtained two optimal combinations of primers that are suitable for performing multi-PCR on these three periodontal pathogens, with amplicon sizes of (197 bp, 316 bp, 226 bp) and (197 bp, 316 bp, 641 bp), respectively. The results also demonstrated that by using multi-PCR, the amplification time can be reduced to as short as 3'48'' for the short-sized amplicons, while for T.f (641 bp), the minimum time required was 8'25''. This work provides an effective way to simultaneously amplify the target genes of P.g, T.d and T.f within a short time, and may promote CF-PCR as a practical tool for point-of-care testing of gingivitis.

Acetic acid denaturing pulsed field capillary electrophoresis for RNA separation.

Based on our previous work of in-capillary denaturing polymer electrophoresis, we present a study of RNA molecular separation up to 6.0 kilo nucleotide by pulsed field CE. This is the first systematic investigation of electrophoresis of a larger molecular mass RNA in linear hydroxyethylcellulose (HEC) under pulsed field conditions. The parameters that may influence the separation performance, e.g. gel polymer concentration, modulation depth and pulse frequency, are analyzed in terms of resolution and mobility. For denaturing and separating RNA in the capillary simultaneously, 2 M acetic acid was added into the HEC polymer to serve as separation buffer. Result shows that (i) in pulsed field conditions, RNA separation can be achieved in a wide range of concentration of HEC polymer, and RNA fragments between 0.3 and 0.6 kilo nucleotide are sensitive to the polymer concentration; (ii) under certain pulsed field conditions, RNA fragments move linearly as the modulation depth increases; (iii) 12.5 Hz is the resonance frequency for RNA reorientation time and applied frequency.

The influence of polymer concentration, applied voltage, modulation depth and pulse frequency on DNA separation by pulsed field CE.

DNA fragments (0.1-10 kbp (kbp, kilo base pair)) separation by square-wave pulsed field CE in hydroxyethylcellulose (HEC, 1300 K) polymer was performed in this work. The effects of polymer concentration, pulse field strength, pulse frequency and modulation depth were investigated. We found that low HEC (about 0.1%) concentration is suitable for the separation of small DNA fragments (<1 kbp), whereas higher HEC concentration (>0.5%) is appropriated for high-mass DNA molecular (>1 kbp) separation. The mobility of DNA fragments is nearly linearly related to average separation voltage under pulsed field conditions. Higher modulation depth is suited to separate the longer DNA fragments and lower modulation depth favors the resolution of short DNA fragments. Thus, the intermediate modulation depth (100%) and pulse frequency (about 31.3 Hz) are prerequisite for high-resolution DNA separation.

Injectable, highly flexible, and thermosensitive hydrogels capable of delivering superoxide dismutase.

Injectable hydrogels are attractive for cell and drug delivery. In this work, we synthesized a family of injectable, biodegradable, fast gelling and thermosensitive hydrogels based on N-isopropylacrylamide (NIPAAm), acrylic acid (AAc), dimethyl-gamma-butyrolactone acrylate (DBA), and 2-hydroxyethyl methacrylate-poly(trimethylene carbontate) (HEMAPTMC) macromer. Type I collagen was composited with the hydrogels to improve their biocompatibility. The hydrogel copolymer solutions were readily injectable at 4 degrees C. The solutions exhibited thermal transition temperatures ranging from 23.6 to 24.5 degrees C and were capable of gelation within 7 s at 37 degrees C to form highly flexible and soft hydrogels with moduli from 39 to 119 KPa and breaking strains >1000%, depending on the copolymer composition and collagen addition. After 2 weeks incubation in PBS, the hydrogels demonstrated weight losses ranging from 10-20%. The completely degraded hydrogels had thermal transition temperatures >40 degrees C and were soluble at body temperature. Superoxide dismutase (SOD) was encapsulated in the hydrogels for the purpose of capturing superoxide within the inflammatory tissue after being delivered in vivo. The hydrogels demonstrated a sustained release profile during a 21-day release period. The release kinetics was dependent on the SOD loading, collagen addition, hydrogel degradation and water content. The released SOD remained bioactive during the entire release period. To test in vitro if the loaded SOD could protect cells encapsulated within the hydrogel from attack by superoxide, human mesenchymal stem cells (MSC) were encapsulated in SOD-loaded hydrogels and cultured in medium containing superoxide generated by activated macrophages. It was found that SOD loading largely suppressed superoxide penetration into the hydrogel and cell membrane. Under normal culture conditions, SOD loading stimulated MSC growth. The SOD-loaded hydrogel exhibited significantly higher cell numbers than the non-SOD loaded hydrogel during a 7-day culture period. These results demonstrated that the developed hydrogels could be used as delivery vehicles for stem cell therapy and drug delivery.

Fabrication and characterization of prosurvival growth factor releasing, anisotropic scaffolds for enhanced mesenchymal stem cell survival/growth and orientation.

Scaffolds that not only mimic the mechanical and structural properties of the target tissue but also support cell survival/growth are likely necessary for the development of mechanically functional cardiovascular tissues. To reach these goals, we have generated scaffolds that are elastic to approximate soft tissue mechanical properties, are nanofibrous to mimic fibrous nature of extracellular matrix (ECM), have aligned structure to guide cellular alignment, and are capable of releasing insulin-like growth factor (IGF-1) to administrate cellular growth and survival. We have developed a technique that can quickly fabricate (<3 h) such scaffolds by simultaneously electrospinning elastase-sensitive polyurethaneurea nanofibers, encapsulating IGF-1 into poly(lactide-co-glycolide) (PLGA) microspheres and assembling them into scaffolds. Scaffold morphology, mechanical properties, degradation with or without elastase, and bioactivity of the released IGF-1 were assessed. The scaffolds had degree of alignment approximately 70%. They were flexible and relatively strong, with tensile strengths of 3.4-11.1 MPa, elongations at break of 71-88%, and moduli of 2.3-7.9 MPa at the alignment direction. IGF-1 release profile and bioactivity were dependent on PLGA content and molecular weight and IGF-1 loading. The released IGF-1 remained bioactive for 4 weeks. The fabricated nanofibers were elastase-sensitive with weight remaining <59% after a 4-week degradation in the presence of elastase. Mesenchymal stem cells (MSCs) were seeded on the scaffolds and cultured either under normal culture conditions (21% O(2), 5% CO(2), and 20% fetal bovine serum (FBS)) or hypoxia/nutrient starvation conditions (5% O(2), 5% CO(2), and 1% FBS) to evaluate the effect of IGF-1 loading on cell growth and survival. Under normal culture conditions, MSCs were found to align on the scaffolds with a degree of alignment matching that of the scaffold. The IGF-1 loaded scaffolds enhanced MSC growth during a 7-day culture period, with higher IGF-1 content showing better stimulus effect. Under hypoxia/nutrient starvation conditions, the IGF-1 loaded scaffolds were found to significantly improve MSC survival.

All-in-one microfluidic device for on-site diagnosis of pathogens based on an integrated continuous flow PCR and electrophoresis biochip.

Current continuous flow polymerase chain reaction (CF-PCR) microfluidic chips require external precision syringe pumps and off-line methods (e.g., electrophoresis and hybridization) to detect PCR products, resulting in complex operations and possible cross-contamination and consequently CF-PCR is still confined to laboratories. Herein, a portable all-in-one microfluidic device is fabricated for rapid diagnosis of pathogens based on an integrated CF-PCR and electrophoresis biochip. A new method was proposed for automatic sample injection into the chip which can substitute the costly external precision syringe pump. It not only achieves rapid DNA amplification and on-site PCR product detection, but also realizes automatic sample injection. As an application, three periodontal pathogens (e.g., Porphyromonas gingivalis, Treponema denticola and Tannerela forsythia) were successfully amplified in the device. Treponema denticola was amplified in as short as 2'31'', and detection of PCR products was completed within 3'43''. The minimum number of bacteria that can be amplified was 125 cfu per µl. The all-in-one device has the potential to be applied in point-of-care nucleic acid testing for diseases.

Capillary electrophoresis of RNA in hydroxyethylcellulose polymer with various molecular weights.

Recent research demonstrates that large numbers of long noncoding RNAs (lncRNAs) in mammals exhibit indices of functionality, and thus analysis of longer RNAs is of great significance. In the present work, we investigated the effect of molecular weight on the separation performance of long RNA by capillary electrophoresis (CE). Results demonstrate that (1) low molecular weight of hydroxyethylcellulose (HEC) (90k) favors the separation of short RNA (<1000 nt). The resolution for short RNA was improved and the migration time was linearly extended with the increase of polymer concentration. (2) In the longer chain HEC (250k, 720k and 1300k), the resolution for the small RNA fragment (<1000 nt) became better as the polymer concentration increased, whereas the resolution for the large ones (>3000 nt) deteriorated. (3) Based on logarithmic plot, there exist two migration regimes for RNA in short chain HEC (90k), three regimes in moderate chain HEC (250k and 720k), and four regimes in the long chain HEC (1300k). Such a systematic investigation of long RNAs may be useful for research on lncRNAs in the length range of 100-10,000 nt.