PicoGreen assay for nucleic acid quantification - Applications, challenges, and solutions.

Various analytical methods and reagents have been employed for nucleic acid analysis in cells, biological fluids, and formulations. Standard techniques like gel electrophoresis and qRT-PCR are widely used for qualitative and quantitative nucleic acid analysis. However, these methods can be time-consuming and labor-intensive, with limitations such as inapplicability to small RNA at low concentrations and high costs associated with qRT-PCR reagents and instruments. As an alternative, PicoGreen (PG) has emerged as a valuable method for the quantitative analysis of nucleic acids. PG, a fluorescent dye, enables the quantitation of double-stranded DNA (dsDNA) or double-stranded RNA, including miRNA mimic and siRNA, in solution. It is also applicable to DNA and RNA analysis within cells using techniques like FACS and fluorescence microscopy. Despite its advantages, PG's fluorescence intensity is affected by various experimental conditions, such as pH, salts, and chemical reagents. This review explores the recent applications of PG as a rapid, cost-effective, robust, and accurate assay tool for nucleic acid quantification. We also address the limitations of PG and discuss approaches to overcome these challenges, recognizing the expanding range of its applications.

Practical magnetic bead-based capillary electrophoresis with laser-induced fluorescence for detecting endogenous miRNA in plasma.

MicroRNAs (miRNAs) are small, non-coding RNAs crucial for gene regulation and implicated in various human diseases. Their potential as clinical prognostic and diagnostic biomarkers in biological fluids necessitates reliable detection methods. In this study, a combination of streptavidin-coupled magnetic beads and capillary electrophoresis with laser-induced fluorescence (CE-LIF) was used to extract and analyze plasma miRNAs. Specifically, miRNAs hybridized with a biotinylated fluorescent DNA probe were isolated from plasma using magnetic beads. These hybridized miRNAs were then directly injected into the CE-LIF system for analysis, eliminating the need for additional processing steps. Both the hybridization and bead-to-probe binding were executed concurrently, regulated by temperature and time. Through the optimization of magnetic bead extraction and CE-LIF conditions, we developed a highly sensitive assay for miR-21 quantification in plasma. The assay displayed remarkable linearity (R2 = 0.9975) within a 0.1-5 pM range and exhibited favorable precision (0.22-1.26 %) and accuracy (98.31-111.19 %). Importantly, we successfully detected endogenous miR-21 in plasma samples from both a lung cancer patient and healthy adults, revealing a 1.7-fold overexpression of miR-21 in lung cancer plasma relative to normal samples. Our findings suggest that this developed system offers a simple and sensitive approach for detecting endogenous miRNAs in plasma, showing its potential utility in disease diagnostics. To our knowledge, this is the first study to utilize CE-LIF for plasma miRNA detection.

Stability Enhancement of Freeze-Dried Gelatin/Alginate Coacervates for bFGF Delivery.

Chronic wound sites have elevated levels of proteolytic enzymes that negate the activity of topically applied growth factors. bFGF encapsulated in gelatin/alginate coacervates was protected from protease and showed better activity than bFGF in solution; however, its activity decreased with particle size and PDI increase after freeze-drying and rehydration. In this study, we aim to improve the stability of bFGF coacervates during freeze-drying to enable a topically applied growth factor delivery system for diabetic foot ulcer. Trehalose, mannitol, and Tween 80 at various concentrations were tested as cryoprotectant candidates. Trehalose improved the mechanical property of freeze-dried coacervates and physical properties after rehydration, resulting in stable size and PDI values. It also enhanced the bFGF activity in hyperglycemic human dermal fibroblasts with better cell viability, migration, and procollagen synthesis compared to the coacervates without trehalose. Hydrogen bonding interactions between trehalose and polymers probed by ATR-FTIR contribute to the stability of coacervates during freeze-drying. In conclusion, the freeze-dried gelatin/alginate coacervates encapsulating bFGF was effectively stabilized with trehalose, and the resulting coacervate composition is suggested as a potential therapeutic modality for chronic wounds including diabetic foot ulcer.

Coacervates: Recent developments as nanostructure delivery platforms for therapeutic biomolecules.

Coacervation is a liquid-liquid phase separation that can occur in solutions of macromolecules through self-assembly or electrostatic interactions. Recently, coacervates composed of biocompatible macromolecules have been actively investigated as nanostructure platforms to encapsulate and deliver biomolecules such as proteins, RNAs, and DNAs. One particular advantage of coacervates is that they are derived from aqueous solutions, unlike other nanoparticle delivery systems that often require organic solvents. In addition, coacervates achieve high loading while maintaining the viability of the cargo material. Here, we review recent developments in the applications of coacervates and their limitations in the delivery of therapeutic biomolecules. Important factors for coacervation include molecular structures of the polyelectrolytes, mixing ratio, the concentration of polyelectrolytes, and reaction conditions such as ionic strength, pH, and temperature. Various compositions of coacervates have been shown to deliver biomolecules in vitro and in vivo with encouraging activities. However, major hurdles remain for the systemic route of administration other than topical or local delivery. The scale-up of manufacturing methods suitable for preclinical and clinical evaluations remains to be addressed. We conclude with a few research directions to overcome current challenges, which may lead to successful translation into the clinic.

Considerations and Suggestions for the Reliable Analysis of miRNA in Plasma Using qRT-PCR.

MicroRNAs (miRNAs) are promising molecules that can regulate gene expression, and their expression level and type have been associated with early diagnosis, targeted therapy, and prognosis of various diseases. Therefore, analysis of miRNA in the plasma or serum is useful for the discovery of biomarkers and the diagnosis of implicated diseases to achieve potentially unprecedented progress in early treatment. Numerous methods to improve sensitivity have recently been proposed and confirmed to be valuable in miRNA detection. Specifically, quantitative reverse-transcription polymerase chain reaction (qRT-PCR) is an effective and common method for sensitive and specific analysis of miRNA from biological fluids, such as plasma or serum. Despite this, the application of qRT-PCR is limited, as it can be affected by various contaminants. Therefore, extraction studies have been frequently conducted to maximize the extracted miRNA amount while simultaneously minimizing contaminants. Moreover, studies have evaluated extraction efficiency and normalization of the extracted sample. However, variability in results among laboratories still exists. In this review, we aimed to summarize the factors influencing the qualification and quantification of miRNAs in the plasma using qRT-PCR. Factors influencing reliable analysis of miRNA using qRT-PCR are described in detail. Additionally, we aimed to describe the importance of evaluating extraction and normalization for reliable miRNA analysis and to explore how miRNA detection accuracy, especially from plasma, can be improved.

Antimicrobial Activities of Propolis in Poloxamer Based Topical Gels.

Propolis contains a group of compounds with various activities. However, their low solubility is a drawback for the development of pharmaceutical formulations. In this study, poloxamers as a solubilizer and gelling agent were evaluated to develop a topical antimicrobial formulation of propolis. The effects of poloxamer type and concentration on the propolis solubility, release rate, and antimicrobial activities were investigated. Staphylococcus aureus (S. aureus) and Candida albicans (C. albicans) were the representative bacteria and fungi, respectively. At 5%, poloxamer 407 (P407) and poloxamer 188 (P188) enhanced the propolis solubility by 2.86 and 2.06 folds, respectively; at 10%, they were 2.81 and 2.59 folds, respectively. The micelle size in the P188 formulation increased in the presence of propolis, whereas there was no change in the P407 formulation. Release rates of propolis decreased with the P188 concentration increase, which was attributed to viscosity increase. Both P188 and P407 formulations showed antimicrobial activity against S. aureus in a time-kill kinetics assay. However, only the P188 formulation reduced the cell's numbers significantly against C. albicans, compared to the control. We speculate that P188 mixed micelles were more effective in releasing free active compounds to exhibit anti-microbial activity compared to the P407 micelles encapsulating the hydrophobic compounds in their cores. Propolis in P188 formulation is proposed as a potential topical antimicrobial agent based on its activity against both S. aureus and C. albicans.

Gelatin-Alginate Coacervates Optimized by DOE to Improve Delivery of bFGF for Wound Healing.

Metabolic disorders in diabetic patients are associated with altered protein and lipid metabolism and defects in granulation tissue formation, resulting in non-healing wounds such as diabetic foot ulcers (DFU). Growth factors have essential roles in tissue re-epithelization and angiogenesis during wound healing. In this study, a complex coacervate was evaluated as an enhanced delivery system for fibroblast growth factor (bFGF) to control its release rate and protect it from proteases. Coacervates composed of gelatin Type A (GA) and sodium alginate (SA) were optimized by the Design of Experiments (DOE), with the polymer ratio and the medium's pH as the independent variables, and turbidity, particle size, polydispersity index, and encapsulation efficiency (EE, %) as the responses. The optimized coacervate protected bFGF from trypsin digestion and showed controlled release compared with bFGF in solution or a physical mixture of GA and SA. It enhanced the viability, migration, and procollagen I C-terminal propeptide synthesis of human dermal fibroblasts in hyperglycemic conditions. In summary, the DOE approach was successfully applied to optimize bFGF GA-SA coacervates as a potential novel therapeutic modality to treat DFU.

Screening of miRNAs in plasma as a diagnostic biomarker for cardiac disease based on optimization of extraction and qRT-PCR condition assay through amplification efficiency.

BACKGROUND: Although quantitative real-time PCR (qRT-PCR) is a common and sensitive method for miRNAs analysis, it is necessary to optimize conditions and minimize qRT-PCR inhibitors to achieve reliable results. The aim of this study was to minimize interference by contaminants in qRT-PCR, maximize product yields for miRNA analyses, and optimize PCR conditions for the reliable screening of miRNAs in plasma. METHODS: The annealing temperature was first optimized by assessing amplification efficiencies. The effects of extraction conditions on levels of inhibitors that interfere with PCR were evaluated. The tested extraction conditions were the volume of the upper layer taken, number of chloroform extractions, and the inclusion of ethanol washing, a process that reduces PCR interference during RNA extraction using TRIzol. RESULTS: An acceptable amplification efficiency of RT-qPCR was achieved by the optimization of the annealing temperature of the tested miRNAs and by the collection a supernatant volume corresponding to about 50% of the volume of TRIzol with triple chloroform extraction. These optimal extraction and PCR conditions were successfully applied to plasma miRNA screening to detect biomarker candidates for the diagnosis of acute myocardial infarction. CONCLUSION: This is the first study to optimize extraction and qRT-PCR conditions, while improving miRNA yields and minimizing the loss of extracted miRNA by evaluations of the amplification efficiency.

Evaluation of Pharmacokinetics and Metabolism of Phosphorothioate Antisense Oligonucleotide G3139 in Rat by Capillary Electrophoresis with Laser-Induced Fluorescence.

A sensitive and specific capillary electrophoresis with laser-induced fluorescence (CE-LIF) and a simple extraction process was developed to simultaneously detect G3139 and its metabolites as a model of antisense oligonucleotides (ASOs). This method has shown excellent linearity within the tested concentration range for G3139 and its metabolites, with a detection limit of 3.0 pM and a recovery of >84.2%. Based on our developed plasma extraction method, we have evaluated the pharmacokinetics and metabolites from rat plasma after intravenous administration of G3139 at 0.76 mg/kg. The results showed that G3139 and its metabolites were successfully simultaneously detected and analyzed through a single run using CE-LIF with baseline separation until the 30-h test sampling time point. The half-life of G3139 and its metabolites was observed at 31 and 68 h, respectively. This study may provide an effective analytical method for the pharmacokinetic and metabolite evaluation required to develop ASOs to treat a variety of diseases.

Solution-Mediated Phase Transformation of Aripiprazole: Negating the Effect of Crystalline Forms on Dissolution and Oral Pharmacokinetics.

We aimed to evaluate the effect of crystalline forms of aripiprazole, an antipsychotic drug for schizophrenia, on the dissolution rates and oral pharmacokinetics. Solubility, intrinsic dissolution rates, and tablet dissolution rates of the monohydrate (MA) and the anhydrous form (AA) were measured in various aqueous media while monitoring the phase transformation by ATR-FTIR. And their oral pharmacokinetics in dogs were compared. The intrinsic dissolution rate of MA was lower compared to AA, confirming its thermodynamic stability relative to AA in water. Phase transformations during the solubility measurement were media-dependent: In simulated gastric fluid, both AA and MA changed to HCl salt form, whereas AA and HCl salt form transformed to MA in simulated intestinal fluid. In vitro dissolution rates and dog oral pharmacokinetics of AA and MA tablets were similar. The results suggest that the solution-mediated transformation to HCl salt or MA negates the effect of different crystalline forms on dissolution rates in vivo and, consequently, on oral pharmacokinetics. We emphasize the importance of the dissolution tests employing various bio-relevant media for better prediction of in vivo performance and the selection of a solid form for development.

Improved Solubility and Oral Absorption of Emodin-Nicotinamide Cocrystal Over Emodin with PVP as a Solubility Enhancer and Crystallization Inhibitor.

Emodin exerts anti-inflammatory and anti-cancer effects. However, its poor water solubility limits development into a pharmaceutical product. Although an emodin-nicotinamide cocrystal (ENC) with improved dissolution rate was proposed as a potential candidate, crystallization back to emodin after dissolution diminished the advantage of the cocrystal approach. The objectives of this study were to identify a crystallization inhibitor to maintain the emodin supersaturation generated by ENC dissolution, and to examine its effect on oral pharmacokinetics of ENC. Among various polymers, polyvinylpyrrolidone K30 (PVP) was the most effective solubilizer and crystallization inhibitor. The solubility of ENC in a simulated intestinal fluid containing 1.5% PVP was 2-fold higher than that of emodin. However, comparison of oral pharmacokinetics in rats between ENC and emodin did not reflect such improved solubility of ENC in vitro relative to emodin. Instead, the plasma concentrations of a major metabolite of emodin showed a positive correlation with in vitro dissolution results, suggesting rapid gastrointestinal metabolism of emodin during absorption. In conclusion, PVP contributes to enhanced dissolution rates of ENC and inhibits crystallization of emodin in vivo, so that more metabolites can be formed and absorbed. Therefore, a metabolism inhibitor would be necessary to improve the oral bioavailability of emodin further.

Improved Diabetic Wound Healing by EGF Encapsulation in Gelatin-Alginate Coacervates.

Topical imageplication of epidermal growth fctor (EGF) has been used to accelerate diabetic foot ulcers but with limited efficacy. In this study, we selected a complex coacervate (EGF-Coa) composed of the low molecular weight gelatin type A and sodium alginate as a novel delivery system for EGF, based on encapsulation efficiency and protection of EGF from protease. EGF-Coa enhanced in vitro migration of keratinocytes and accelerated wound healing in streptozotocin-induced diabetic mice with increased granulation and re-epithelialization. While diabetic wound sites without treatment showed downward growth of hyperproliferative epidermis along the wound edges with poor matrix formation, EGF-Coa treatment recovered horizontal migration of epidermis over the newly deposited dermal matrix. EGF-Coa treatment also resulted in reduced levels of proinflammatory cytokines IL-1, IL-6, and THF-α. Freeze-dried coacervates packaged in aluminum pouches were stable for up to 4 months at 4 and 25 °C in terms of appearance, purity by RP-HPLC, and in vitro release profiles. There were significant physical and chemical changes in relative humidity above 33% or at 37 °C, suggesting the requirement for moisture-proof packaging and cold chain storage for long term stability. We propose low molecular weight gelatin type A and sodium alginate (LWGA-SA) coacervates as a novel EGF delivery system with enhanced efficacy for chronic wounds.

A rapid and reliable CE-LIF method for the quantitative analysis of miRNA-497 in plasma and organs and its application to a pharmacokinetic and biodistribution study.

MicroRNAs (miRNAs) are involved in the pathogenesis of many human diseases, and various miRNAs have been reported and developed as therapeutic candidates for treating various diseases. Various miRNA and carrier modification systems have been investigated for effective systemic miRNA delivery to cells, organs, and tissues of interest. Consequently, effective and reliable analytical methods of miRNAs are required for evaluating the pharmacokinetics and biodistribution of miRNAs as therapeutic candidates. The capillary electrophoresis with laser-induced fluorescence (CE-LIF) method has been recently reported as a promising and relatively rapidly developing tool with the potential to provide highly sensitive and specific analysis of biological molecules including miRNAs. Here, the CE-LIF method was used for application in the pharmacokinetic and distribution studies of miRNA-497 as a model miRNA for a lung target; miRNA-497 hybridized with 6-FAM-labeled DNA probes were separated using CE-LIF and detected within 6 min without any interference. This method showed a wide calibration range of 1.0-50 nM and 0.1-50 nM for plasma and the four organs, liver, spleen, lung, and kidney, respectively, with acceptable precision and accuracy. Using CE-LIF, the miRNA-497 level was evaluated in rat plasma and organs after intravenously administering 1 mg ml-1 of a miRNA-497 mimic. Hence, miRNA-497 displayed a relatively short half-life of 1.76 h and was delivered to the lungs but mainly accumulated in the liver and spleen. This study evaluated the pharmacokinetics and biodistribution of the miRNA-497 mimic using CE-LIF for the first time and suggested the need for further studies to extend the half-life and conduct lung-targeted delivery of miRNA-497.

Accelerated wound healing in diabetic mice by miRNA-497 and its anti-inflammatory activity.

Diabetic foot ulcers represent one of the major and rising health issues, as the number of diabetic patients is increasing. MicroRNAs (miRNAs) are among various bioactive molecules under investigation for diabetic wound healing. The prolonged pro-inflammatory phase in diabetic wounds partly attributes to its non-healing nature. Therefore, we hypothesized that miRNA-497, known for its regulation of inflammatory responses, would enhance diabetic wound healing. We screened miRNA candidates, including miRNA-497 in the wounded skin of streptozotocin-induced type 1 diabetic mice. The therapeutic potential of miRNA-497 mimic was studied by intradermal injection around the wound in diabetic mice. In addition, the effects of miRNA-497 on pro-inflammatory cytokines were analyzed in the wound lesion of diabetic mice, and in human dermal fibroblasts cells exposed to high glucose and lipopolysaccharide.We found a significant reduction of miRNA-497 expression in the dermal wounds of the diabetic mice relative to normal mice. Intradermal injection of miRNA-497 around the full-thickness dermal wounds in diabetic mice accelerated wound closure effectively compared to the control miRNA. miRNA-497 treatment in vivo and in vitro decreased representative pro-inflammatory cytokines such as IL-1ß, IL-6, and TNF-α. Such anti-inflammatory effects of miRNA-497 shed light on its role in accelerating diabetic wound healing. In conclusion, miRNA-497, with its down-regulation activity for pro-inflammatory cytokines, is proposed as a potential therapeutic agent for diabetic wound healing.

MiR-195 and miR-497 suppress tumorigenesis in lung cancer by inhibiting SMURF2-induced TGF-ß receptor I ubiquitination.

SMURF2 is a member of the HECT family of E3 ubiquitin ligases that have important roles as a negative regulator of transforming growth factor-ß (TGF-ß) signaling through ubiquitin-mediated degradation of TGF-ß receptor I. However, the regulatory mechanism of SMURF2 is largely unknown. In this study, we identified that micro(mi)R-195 and miR-497 putatively target SMURF2 using several target prediction databases. Both miR-195 and miR-497 bind to the 3'-UTR of the SMURF2 mRNA and inhibit SMURF2 expression. Furthermore, miR-195 and miR-497 regulate SMURF2-dependent TßRI ubiquitination and cause the activation of the TGF-ß signaling pathway in lung cancer cells. Upregulation of miR-195 and miR-497 significantly reduced cell viability and colony formation through the activation of TGF-ß signaling. Interestingly, miR-195 and miR-497 also reduced the invasion ability of lung cancer cells when cells were treated with TGF-ß1. Subsequent in vivo studies in xenograft nude mice model revealed that miR-195 and miR-497 repress tumor growth. These findings demonstrate that miR-195 and miR-497 act as a tumor suppressor by suppressing ubiquitination-mediated degradation of TGF-ß receptors through SMURF2, and suggest that miR-195 and miR-497 are potential therapeutic targets for lung cancer.

Delivery of therapeutic miRNA using polymer-based formulation.

MicroRNAs (miRNAs) are short non-coding RNAs that play important roles in many cellular processes such as development, proliferation, differentiation, and apoptosis. For this reason, miRNAs have been proposed and investigated as biomarkers and therapeutics for various diseases such as cancer, diabetes, and cardiovascular disease. However, delivery of miRNAs and their antagomirs to target sites remains challenging because of poor cellular uptake and degradation by nucleases. Various delivery systems have been investigated for enhanced delivery of miRNAs to cells, organs, and tissues of interest, thereby enabling evaluation of their biological functions and clinical trials. In particular, non-viral, polymer-based carriers have shown advantages such as versatility of structural modifications and protection of unstable miRNA. Herein, we review properties and applications of poly (lactic-co-glycolic acid), chitosan, polyethyleneimine, and polyamidoamine dendrimers as carriers for effective delivery systems of miRNA mimic or anti-miRNAs that directly target essential miRNAs and/or their target genes. A number of miRNAs in clinical trials appear to use chemically modified miRNAs without any particular delivery system except one study with liposomal miRNA. With more accumulation of positive research results on polymeric delivery of miRNA in vitro and in vivo, we expect that polymeric delivery system will accelerate advancement of miRNA therapeutics to clinical study in the near future.

Emodin-nicotinamide (1:2) cocrystal identified by thermal screening to improve emodin solubility.

Emodin (EM), an anthraquinone obtained from natural products, is known for many pharmacological activities. However, further evaluation and interpretation of toxicity or pharmacological activity of emodin are limited due to its poor aqueous solubility. We aimed to identify an emodin cocrystal with improved pharmaceutical properties. Among various compounds screened by thermal analysis, nicotinamide (NCT) was identified as a potential cocrystal coformer, based on the presence of an exothermal peak in DSC profiles of the physical mixture of EM and NCT. Crystallization of EM-NCT cocrystal (EM-NCT) using slow or rapid solvent evaporation method yielded a novel cocrystal at 1:2 ratio. Single crystal structure analysis revealed EM dimers and NCT tetramers connected alternatively via H-bonds to make one-dimensional chains which are joined by inter-chain H-bonds between NCT to form two-dimensional layers. The EM molecules are planar with intramolecular H-bonds between O atoms. Compared with EM, the EM-NCT cocrystal showed improved aqueous solubility, dissolution rate, and stability. Hence, EM-NCT cocrystal is proposed as a more suitable solid form for further development as pharmaceutical products.

Neutrophil elastase inhibition effectively rescued angiopoietin-1 decrease and inhibits glial scar after spinal cord injury.

After spinal cord injury (SCI), neutrophil elastase (NE) released at injury site disrupts vascular endothelium integrity and stabilization. Angiopoietins (ANGPTs) are vascular growth factors that play an important role in vascular stabilization. We hypothesized that neutrophil elastase is one of the key determinants of vascular endothelium disruption/destabilization and affects angiopoietins expression after spinal cord injury. To test this, tubule formation and angiopoietins expression were assessed in endothelial cells exposed to different concentrations of recombinant neutropil elastase. Then, the expression of angiopoietin-1, angiopoietin-2, and neutrophil elastase was determined at 3 h and at 1, 3, 5, 7, 14, 21, and 28 days in a clinically relevant model of moderate compression (35 g for 5 min at T10) spinal cord injury. A dichotomy between the levels of angiopoietin-1 and angiopoietin-2 was observed; thus, we utilized a specific neutrophil elastase inhibitor (sivelestat sodium; 30 mg/kg, i.p., b.i.d.) after spinal cord injury. The expression levels of neutropil elastase and angiopoietin-2 increased, and that of angiopoietin-1 decreased after spinal cord injury in rats. The sivelestat regimen, optimized via a pharmacokinetics study, had potent effects on vascular stabilization by upregulating angiopoietin-1 via the AKT pathway and preventing tight junction protein degradation. Moreover, sivelestat attenuated the levels of inflammatory cytokines and chemokines after spinal cord injury and hence subsequently alleviated secondary damage observed as a reduction in glial scar formation and the promotion of blood vessel formation and stabilization. As a result, hindlimb locomotor function significantly recovered in the sivelestat-treated animals as determined by the Basso, Beattie, and Bresnahan scale and footprint analyses. Furthermore, sivelestat treatment attenuated neuropathic pain as assessed by responses to von Frey filaments after spinal cord injury. Thus, our result suggests that inhibiting neutropil elastase by administration of sivelestat is a promising therapeutic strategy to inhibit glial scar and promote functional recovery by upregulating angiopoietin-1 after spinal cord injury.

An improvement of miRNA extraction efficiency in human plasma.

MicroRNAs (miRNAs) are short noncoding RNA molecules that control the expression of mRNAs associated with various biological processes. Therefore, deregulated miRNAs play important roles in the pathogenesis of diseases. Numerous studies are aimed at discovering biomarkers of diseases or determining miRNA functions by monitoring circulating miRNAs in various biological sources such as plasma and urine. However, the analysis of miRNA in such fluids presents problems related to accuracy and reproducibility because of their low levels in biological fluids. Therefore, better extraction kits and more sensitive detection systems have been developed for improved and reproducible analysis of circulating miRNAs. However, new extraction methods are also needed to improve the yield of miRNAs for their reliable analysis from biological fluids. The combination of yeast transfer RNA (tRNA) and glycogen as carrier molecules and incubation durations were optimized to maximize extraction efficiency. The extraction recovery using a combination of yeast tRNA and glycogen was approximately threefold more than that by using glycogen or yeast tRNA alone. In addition, reproducible and accurate analysis of miRNAs can be carried out after extraction using a combination of yeast tRNA and glycogen without an impact on plasma components. Graphical abstract Steps of miRNA extraction in plasma.

MIR-27a regulates the TGF-ß signaling pathway by targeting SMAD2 and SMAD4 in lung cancer.

The transforming growth factor-ß (TGF-ß) signaling pathway is associated with carcinogenesis and various biological processes. SMAD2 and SMAD4, which are putative tumor suppressors, have an important role in TGF-ß signaling. The aberrant expression of these genes is implicated in some cancers. However, the mechanisms of SMAD2 and SMAD4 dysregulation are poorly understood. In this study, we observed that miR-27a was upregulated in lung cancer cell lines and patients. In addition, SMAD2 and SMAD4 genes were identified as targets of miR-27a by several target prediction databases and experimental validation. Functional studies revealed that miR-27a overexpression decreased SMAD2 and SMAD4 mRNA and protein levels. Furthermore, miR-27a contributed to cell proliferation and invasion by inhibiting TGF-ß-induced cell cycle arrest. These results suggest that miR-27a may function as an oncogene by regulating SMAD2 and SMAD4 in lung cancer. Thus, miR-27a may be a potential target for cancer therapy.