Solvatochromic peptidic binder obtained via extended phage display acts as a fluororeporter for fragment-based drug discovery (FBDD).

We have previously established a selection system to obtain a solvatochromic protein binder from a peptidic fluoroprobe library via the extended T7 phage display. Here, we use the peptidic binder as a fluororeporter in this proof-of-concept study of fragment-based screening approach to drug discovery. The binder is released from the target protein on mixing with an appropriate lead compound, thereby altering its fluorescence color/intensity under 365 nm ultraviolet wavelength irradiation. By this instant screening outcome, the affinity of the lead compound is apparent to the naked eye, and quantified with a portable microvolume fluorophotometer. We envision that our simple and affordable screening system will provide opportunities for early stage drug discovery, especially for non-experts in academia and education because expensive hardware is not required for qualifying the measurements.

Comparison of five assays for DNA extraction from bacterial cells in human faecal samples.

AIM: To determine the most effective DNA extraction method for bacteria in faecal samples. MATERIALS AND RESULTS: This study assessed five commercial methods, that is, NucliSens easyMag, QIAamp DNA Stool Mini kit, PureLink Microbiome DNA purification kit, QIAamp PowerFecal DNA kit and RNeasy PowerMicrobiome kit, of which the latter has been optimized for DNA extraction. The DNA quantity and quality were determined using Nanodrop, Qubit and qPCR. The PowerMicrobiome kit recovered the highest DNA concentration, whereby this kit also recovered the highest gene copy number of Gram positives, Gram negatives and total bacteria. Furthermore, the PowerMicrobiome kit in combination with mechanical pre-treatment (bead beating) and with combined enzymatic and mechanical pre-treatment (proteinase K+mutanolysin+bead beating) was more effective than without pre-treatment. CONCLUSION: From the five DNA extraction methods that were compared, the PowerMicrobiome kit, preceded by bead beating, which is standard included, was found to be the most effective DNA extraction method for bacteria in faecal samples. SIGNIFICANCE AND IMPACT OF THE STUDY: The quantity and quality of DNA extracted from human faecal samples is a first important step to optimize molecular methods. Here we have shown that the PowerMicrobiome kit is an effective DNA extraction method for bacterial cells in faecal samples for downstream qPCR purpose.

Study on the admission levels of circulating cell-free DNA in patients with acute myocardial infarction using different quantification methods.

Circulating cell-free DNA (cf-DNA) is present in human biological fluids, mainly in plasma and serum, originating from cell death, a process that massively takes place during acute myocardial infarction (AMI). In the present study, cf-DNA was assessed by different quantification techniques, in order to determine its levels in patients admitted with AMI. A total of 130 subjects were included in the study: 80 ST elevation myocardial infarction (STEMI) patients and 50 healthy controls. Cf-DNA extracted from plasma was analyzed by: a) Qubit 3.0 with single (ss) and double (ds) stranded DNA assay kits, b) NanoDrop and c) quantitative PCR (qPCR). Cf-DNA levels were recorded elevated in AMI patients compared to those of healthy individuals. Specifically, Qubit 3.0 ss-DNA kit provided the highest cf-DNA concentration values for all the samples analyzed in comparison with ds-DNA assay kit and NanoDrop, approaching the values obtained by qPCR. Cf-DNA augments in massive cell death settings, including AMI, proposing that the quantification of its levels by novel methodologies could contribute to patient diagnosis and clinical management.

Detection of phenol contamination in RNA samples and its impact on qRT-PCR results.

Residual phenol, carried over from RNA purification, can alter RNA concentration measurements and is assumed to inhibit PCR. Here, we demonstrate that Impurities A260 values of spectral content profiling (SCP) UV/Vis measurements correlated with phenol concentration, whereas absorbance ratios of classical UV/Vis systems failed to reliably detect phenol in RNA samples. Phenol contamination led to over- or underestimation of RNA concentration on UV/Vis systems, whereas it had no influence on fluorometry quantification. Wrong RNA concentration results led to altered template input in qRT-PCR and consequently caused quantification cycle (Cq) shifts, although ≤ 0.01% phenol had no direct influence.

Comparative assessment of DNA extraction procedures for Ascaris spp. eggs.

A central and critical step in the molecular detection of soil-transmitted helminths from environmental sources is the extraction of DNA from the eggs. In this study, we investigated the yield of DNA extracted from known quantities (500, 100, 50, 20, 10 and 5) of Ascaris suum eggs, as well as directly from wastewater and sludge samples containing Ascaris spp. eggs, using six commercial DNA extraction kits. The amount of DNA extracted was quantified with NanoDrop, Qubit and Ct values from quantitative polymerase chain reaction (qPCR) assay using CFX96 Touch™ real-time PCR equipment. The PowerLyzer Ultraclean Microbial DNA isolation kit and PowerSoil DNA isolation kit gave the highest yield of DNA based on the NanoDrop, Qubit and Ct values. However, the qPCR results indicate that in some of the kits, PCR inhibitors may have been carried over to the PCR reaction. DNA extraction kits that incorporate a bead-beating step as well as other mechanical eggshell disruption steps were superior in extracting DNA from Ascaris spp. eggs. Additionally, for the accurate quantification of extracted DNA, the use of Ct values from qPCR and Qubit readings gives better results compared to the NanoDrop readings. For efficient downstream applications, the use of DNA extraction kits with superior inhibitor removal technology is essential, in addition to a high yield of DNA.

Correction for concentration overestimation of nucleic acids with phenol.

We report a computational method based on ultraviolet (UV) spectra for correcting the overestimated concentrations of nucleic acid samples contaminated with TRIzol/phenol. The derived correction formulas were validated using RNA solutions, double-stranded DNA solutions, and single-stranded oligonucleotide solutions. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) with SYBR Green was performed to assess the level of TRIzol contamination that can be tolerated for gene expression quantification. After the correction, the accuracy of the RNA concentrations was greatly improved and there was no significant difference in the threshold cycle (Ct) values for GAPDH and ACAN genes in RT-qPCR obtained for RNA contaminated with up to 0.1% TRIzol (phenol level index [PLI]∼5.8-5.9). Similarly, accuracy improvements were also observed for DNA or oligonucleotides contaminated with phenol using different concentration correction formulas. In addition, the Ct values and amplification efficiency of DNA in qPCR were not affected by TRIzol contamination below 1%. This computational method is easy and convenient to use and reduces the concentration overestimations greatly.

An Optimized and Cost-Effective RNA Extraction Method for Secondary Metabolite-Enriched Tissues of Norway Spruce (Picea abies).

Since the development of next-generation sequencing techniques and with the growing interest in transcriptomic studies, there is a demand for high-throughput RNA extraction techniques. General RNA extraction protocols are unreliable when it comes to the quality and quantity of isolated RNA obtained from different tissue types of different plant species. Despite Norway spruce (Picea abies) being one of the most significant and commercially valuable tree species in European forests, only limited genetic research is available. In this study, we developed a cetyltrimethylammonium bromide (CTAB) protocol by modifying the original method. We compared this CTAB protocol with other widely used methods for extracting RNA from different tissues (needle, phloem, and root) of Norway spruce, known for its richness in polyphenols, polysaccharides, and secondary metabolites. The modified CTAB method proves to be superior to the kit-based and TRIzol-based methods for extracting RNA from the metabolite-rich tissues of Norway spruce, resulting in high RNA quality and integrity values (RIN~7-9). The modified CTAB RNA extraction method is rapid, cost-effective, and relatively simple in yielding the desired RNA quality from Norway spruce tissues. It is optimal for RNA sequencing and other downstream molecular applications.

Assessment of measurement accuracy of amplified DNA using a colorimetric loop-mediated isothermal amplification assay.

A colorimetric loop-mediated isothermal amplification assay detects changes in pH during amplification based on color changes at a constant temperature. Currently, various studies have focused on developing and assessing molecular point-of-care testing instruments. In this study, we evaluated amplified DNA concentrations measured using the colorimetric LAMP assay of the 1POT™ Professional device (1drop Inc, Korea). Results of the 1POT analysis of clinical samples were compared with measurements obtained from the Qubit™ 4 and NanoDrop™ 2000 devices (both from Thermo Fisher Scientific, MA, USA). These results showed a correlation of 0.98 (95% CI: 0.96-0.99) and 0.96 (95% CI: 0.92-0.98) between 1POT and the Qubit and NanoDrop. 1POT can measure amplified DNA accurately and is suitable for on-site molecular diagnostics.

Low-cost equilibrium unfolding of heme proteins using 2 µl samples.

Equilibrium unfolding experiments provide access to protein thermodynamic stability revealing basic aspects of protein structure-function relationships. A limitation of these experiments stands on the availability of large amounts of protein samples. Here we present the use of the NanoDrop for monitoring guanidinium chloride-induced unfolding by Soret absorbance of monomeric heme proteins. Unfolding experiments using 2 µl of reactant are validated by fluorescence and circular dichroism spectroscopy and supported with five heme proteins including neuroglobin, cytochrome b5, and cyanoglobin. This work guarantees 2 orders of magnitude reduction in protein expense. Promising low-cost protein unfolding experiments following other chromophores and high-throughput screenings are discussed.

Unveiling the arcanum of formalin-fixed paraffin-embedded archival tissue blocks: A valuable resource for genomic DNA extraction.

Background: Formalin-fixed paraffin-embedded (FFPE) tissue blocks are routinely preserved after pathological diagnosis and possess tremendous potential for biomarker discovery. These archival samples are prone to degradation on prolonged storage due to the formalin cross-linking. Aims: This study aimed to evaluate whether the storage period of the formalin-fixed paraffin-embedded tumor blocks had a significant impact on the yield and purity of the isolated DNA archived for 11 years. Settings and Design: A retrospective study was carried out in the Department of Oral Pathology and Microbiology in accordance with the Institutional Ethics Committee. Materials and Methods: Genomic DNA extraction was performed using TaKaRa DEXPAT Easy DNA kit from 40 FFPE tissue blocks of oral squamous cell carcinoma archived for 11 years (2006-2017). NanoDrop spectrophotometer was used to determine the DNA yield (A260) and purity (A260/A280 ratio). The quality of DNA fragments was validated using agarose gel electrophoresis. Statistical Analysis Used: Statistical analysis was obtained by SPSS 22, MS Excel and analyzed using the analysis of variance (ANOVA) test. P < 0.05 was set for statistical significance. Results: There was no statistically significant difference observed both in terms of DNA yield (P = 0.996) and purity (P = 0.997) of FFPE tumor blocks archived for 11 years among the study groups. Conclusions: It was concluded that, irrespective of years of storage of the FFPE, it is possible to extract genomic DNA and use it for molecular studies.

A simple approach for effective shearing and reliable concentration measurement of ultra-high-molecular-weight DNA.

Pipetting and concentration measurement of viscous ultra-high-molecular-weight (UHMW) DNA samples is challenging and often highly imprecise. Effective guidelines for handling UHMW samples are missing in the field. Herein, a simple and low-cost workflow is presented that enables accurate pipetting and reliable concentration measurement. Central to the workflow is the shearing of representative small aliquots of UHMW DNA samples to a fragment size <150 kb by vortexing them for 1 min with a glass bead in a round-bottomed 2-ml tube. Additionally, a solution is provided for accurate quantitation of high-molecular-weight DNA with fluorometric (Qubit [Thermo Fisher Scientific, MA, USA]) methods by using an appropriate genomic DNA standard, resulting in values that match spectrophotometric (Nanodrop [Thermo Fisher Scientific]) optical density readings.

RNA Isolation from Articular Cartilage Tissue.

Isolation of high-quality RNA directly from tissues is desirable to obtain precise information of in vivo gene expression profiles in cells embedded within their extracellular matrix (ECM). It is well known that purification of RNA from cartilage tissues is particularly challenging due to low cell (chondrocyte) content and its dense ECM rich in large negatively charged proteoglycans that can copurify with RNA. Older methodologies to purify RNA from cartilage involved the use of concentrated denaturing solutions containing guanidinium isothiocyanate followed by ultracentrifugation in cesium trifluoroacetate. Such ultracentrifugation approaches are rarely used now since the emergence of more user-friendly mini spin column chromatography kits. For this chapter, we tested and compared three methods to isolate RNA from immature murine articular (femoral head) cartilage and found that the combination of TRIzol® reagent and spin column chromatography (Norgen Total RNA Purification Kit) was the best approach to generate higher quality RNA. Here, the average RNA Integrity Number (RIN), as determined by Bioanalyzer technology, was 7.1. We then applied this method to attempt to isolate RNA directly from human articular cartilage harvested from three osteoarthritic (OA) knee joint specimens. As expected, the concentration and quality of RNA obtained differed between samples. However, from one specimen, we were able to isolate approximately 3 µg of total RNA (including small noncoding RNAs) from 100 mg of human OA cartilage with a RIN = 7.9. Despite the patient-to-patient variabilities that are known to exist between cartilage specimens from OA joints, we have demonstrated that it is possible to obtain reasonably high levels of RNA from human OA articular cartilage at a quality suitable for downstream analyses including microarray and RNA-Seq. A detailed description of our preferred RNA purification methodology, which can be used to isolate RNA from human, bovine, or rodent cartilage tissue, is provided in this chapter.

Protein Immobilization on Gold Nanoparticles: Quantitative Analysis.

Conjugation of gold nanoparticles (AuNPs) with biologically relevant molecules underpins many applications in medicine and biochemistry. Immobilization of functional proteins on AuNPs often affects protein structure and function. Such effects are protein dependent and require thorough investigation using suitable quantitative tests. Good experimental design and the use of a comprehensive set of control samples are essential when characterizing the consequences of protein immobilization and its effect on protein structure and function. However, traditional approaches to making control samples, that is, immobilized protein versus protein in solution in absence of any nanoparticles, do not provide sufficiently identical reaction conditions and complicate interpretation of the results. Accurate quantification of protein conjugation to AuNPs and ensuring complete removal of unconjugated protein remain the two key challenges in such functional assays. This report describes a simple and straightforward procedure allowing for quantitative analysis of protein conjugation to AuNPs. The principles are illustrated using fluorescence and circular dichroism measurements, and can be applied to other analytical techniques or be adapted with minor modifications for use with other proteins.

Improved DNA extraction and purification with magnetic nanoparticles for the detection of methicillin-resistant Staphylococcus aureus.

Molecular methods offer fast, safe and cost-efficient detection of pathogenic bacteria, including methicillin-resistant Staphylococcus aureus (MRSA). These tests depend on a rapid extraction of bacterial DNA. The aim of this study was to compare an optimized DNA extraction and purification protocol for MRSA using magnetic nanoparticles with the original method. The purity of the extracted DNA was assessed by photometric measurements and the amount of DNA was determined by real-time PCR. Three MRSA reference strains (S. aureus ATCC® 70699, S. aureus ATCC® 43300; S. aureus ATCC® 33592) and eleven MRSA field strains, which include SCCmec elements of types I to XI, were used in this study. The optimized protocol can save approximately 20 min time compared to the original method and the DNA yield was higher with the new protocol. Therefore, this new protocol allows a faster DNA extraction from MRSA cultures.

The value of fluorimetry (Qubit) and spectrophotometry (NanoDrop) in the quantification of cell-free DNA (cfDNA) in malignant melanoma and prostate cancer patients.

BACKGROUND: Circulating cell-free tumor DNA (cfDNA) is of crucial interest in oncology. cfDNA constitutes a potential prognostic and therapeutic marker for different solid tumors and can be used in the diagnostic and therapeutic management of cancer patients for which nowadays there are no valid laboratory markers. In the present study, the quality and quantity of the cfDNA were assessed by different quantification procedures, in order to identify the potential applications of these techniques in the preliminary cfDNA quantification. METHODS: Qubit with single (ss) and double strand (ds) DNA assay kits, NanoDrop and quantitative Real Time PCR (qPCR), were adopted to assess the cfDNA in the blood samples of 18 melanoma patients, 67 prostate cancer patients and 15 healthy controls. RESULTS: The quantification by NanoDrop (average value 8.48ng/µl, 95% confidence limit (CL)=7.23-9.73), Qubit ssDNA (average value 23.08ng/µl, CL=19.88-26.28), dsDNA (average value 4.32ng/µl, CL=3.52-5.12) assay kits and qPCR (average value 0.39ng/µl, CL=0.31-0.47) revealed differences among the four procedures. Qubit 2.0 ss-DNA kit gave higher cfDNA concentration values for all the samples analyzed. In detail, Qubit ssDNA assay revealed higher sensitivity in the quantification of small amounts of pure ss-DNA and ds-DNA, while NanoDrop allowed the assessment of the purity of cfDNA samples. CONCLUSIONS: The NanoDrop and Qubit 2.0 measurements were analyzed in order to define their correlation with qPCR cfDNA assessment, showing good correlation values with the qPCR that should be considered the "gold standard". In our proposal, the sequential combination of NanoDrop and Qubit ssDNA methods should be adopted for a cost-effective preliminary assessment of total circulating cfDNA in melanoma and prostate cancer patients, and only discordant values should undergo qPCR assessment.

Assessment of morphological changes and DNA quantification: An in vitro study on acid-immersed teeth.

CONTEXT: Acid immersion of victim's body is one of the methods employed to subvert identification of the victim, and hence of the perpetrator. Being hardest and chemically the most stable tissue in the body, teeth can be an important forensic investigative medium in both living and nonliving populations. Teeth are also good reservoirs of both cellular and mitochondrial DNA; however, the quality and quantity of DNA obtained varies according to the environment the tooth has been subjected to. DNA extraction from acid-treated teeth has seldom been reported. AIMS: The objectives of the present study were to assess the morphological changes along with DNA recovery from acid-immersed teeth. MATERIALS AND METHODS: Concentrated hydrochloric acid, nitric acid, and sulfuric acid were employed for tooth decalcification. DNA was extracted on an hourly basis using phenol-chloroform method. Quantification of extracted DNA was done using a spectrophotometer. RESULTS: Results showed that hydrochloric acid had more destructive capacity compared to other acids. CONCLUSION: Sufficient quantity of DNA was obtainable till the first 2 hours of acid immersion and there was an inverse proportional relation between mean absorbance ratio and quantity of obtained DNA on an hourly basis.

Explanatory chapter: nucleic acid concentration determination.

This protocol describes a method for determining the concentration of a nucleic acid sample.