An improved method for isolation of RNA from rat femur

Background: RNA isolation from ossified bone is a difficult and time-consuming process which often results in poor recovery of RNA. The yield is limited and might not be suitable for gene quantification studies by real time PCR. Methodology: The present study demonstrates RNA extraction from rat femur utilizing the silica column along with the trizol reagent. Quality of RNA was assessed by agarose gel analysis and its suitability for real-time PCR analysis was determined by β-actin Ct values. Results: The RNA isolated using silica columns in conjugation with trizol reagent resulted in higher yield of RNA and purity (A260/280=2.04; yield =1545.73 µg/ml) compared to the trizol method alone (A260/280=1.85; yield =571.2 µg/ml). Ct value of β actin obtained from RNA isolated by trizol method was higher than the Ct value obtained by trizol in conjugation with the column method (31.41 and 15.41 respectively). Conclusion: Combination of trizol along with silica column resulted in better quality and improved yield of RNA suitable for gene quantification by Real time PCR.     

Demonstration of preferential binding of SYBR Green I to specific DNA fragments in real-time multiplex PCR

SYBR Green I (SG) is widely used in real-time PCR applications as an intercalating dye and is included in many commercially available kits at undisclosed concentrations. Binding of SG to double-stranded DNA is non-specific and additional testing, such as DNA melting curve analysis, is required to confirm the generation of a specific amplicon. The use of melt curve analysis eliminates the necessity for agarose gel electrophoresis because the melting temperature (T_m) of the specific amplicon is analogous to the detection of an electrophoretic band. When using SG for real-time PCR multiplex reactions, discrimination of amplicons should be possible, provided the T_m values are sufficiently different. Real-time multiplex assays for Vibrio cholerae and Legionella pneumophila using commercially available kits and in-house SG mastermixes have highlighted variability in performance characteristics, in particular the detection of only a single product as assessed by T_m analysis but multiple products as assessed by agarose gel electrophoresis. The detected T_m corresponds to the amplicon with the higher G+C% and larger size, suggesting preferential binding of SG during PCR and resulting in the failure to detect multiple amplicons in multiplex reactions when the amount of SG present is limiting. This has implications for the design and routine application of diagnostic real-time PCR assays employing SG.     

Evaluation of conventional and four real-time PCR methods for the detection of Leishmania on field-collected samples in Ethiopia

In most low-resource settings, microscopy still is the standard method for diagnosis of cutaneous leishmaniasis, despite its limited sensitivity. In Ethiopia, the more sensitive molecular methods are not yet routinely used. This study compared five PCR methods with microscopy on two sample types collected from patients with a suspected lesion to advise on optimal diagnosis of Leishmania aethiopica. Between May and July 2018, skin scrapings (SS) and blood exudate from the lesion spotted on filter paper (dry blood spot, DBS) were collected for PCR from 111 patients of four zones in Southern Ethiopia. DNA and RNA were simultaneously extracted from both sample types. DNA was evaluated by a conventional PCR targeting ITS-1 and three probe-based real-time PCRs: one targeting the SSU 18S rRNA and two targeting the kDNA minicircle sequence (the ‘Mary kDNA PCR’ and a newly designed ‘LC kDNA PCR’ for improved L. aethiopica detection). RNAs were tested with a SYBR Green-based RT-PCR targeting spliced leader (SL) RNA. Giemsa-stained SS smears were examined by microscopy. Of the 111 SS, 100 were positive with at least two methods. Sensitivity of microscopy, ITS PCR, SSU PCR, Mary kDNA PCR, LC kDNA PCR and SL RNA PCR were respectively 52%, 22%, 64%, 99%, 100% and 94%. Microscopy-based parasite load correlated well with real-time PCR Ct-values. Despite suboptimal sample storage for RNA detection, the SL RNA PCR resulted in congruent results with low Ct-values. DBS collected from the same lesion showed lower PCR positivity rates compared to SS. The kDNA PCRs showed excellent performance for diagnosis of L. aethiopica on SS. Lower-cost SL RNA detection can be a complementary high-throughput tool. DBS can be used for PCR in case microscopy is negative, the SS sample can be sent to the referral health facility where kDNA PCR method is available.     

Performance of Multiplex Commercial Kits to Quantify Cytokine and Chemokine Responses in Culture Supernatants from Plasmodium falciparum Stimulations

Background

Cytokines and chemokines are relevant biomarkers of pathology and immunity to infectious diseases such as malaria. Several commercially available kits based on quantitative suspension array technologies allow the profiling of multiple cytokines and chemokines in small volumes of sample. However, kits are being continuously improved and information on their performance is lacking.

Methodology/Principal Findings

Different cytokine/chemokine kits, two flow cytometry-based (eBioscience® FlowCytomix™ and BD™ Cytometric Bead Array Human Enhanced Sensitivity) and four Luminex®-based (Invitrogen™ Human Cytokine 25-Plex Panel, Invitrogen™ Human Cytokine Magnetic 30-Plex Panel, Bio-Rad® Bio-Plex Pro™ Human Cytokine Plex Assay and Millipore™ MILLIPLEX® MAP Plex Kit) were compared. Samples tested were supernatants of peripheral blood mononuclear cells of malaria-exposed children stimulated with Plasmodium falciparum parasite lysates. Number of responses in range that could be detected was determined and reproducibility of duplicates was evaluated by the Bland-Altman test. Luminex® kits performed better than flow cytometry kits in number of responses in range and reproducibility. Luminex® kits were more reproducible when magnetic beads were used. However, within each methodology overall performance depended on the analyte tested in each kit. Within the Luminex® kits, the Invitrogen™ with polystyrene beads had the poorer performance, whereas Invitrogen™ with magnetic beads had the higher percentage of cytokines/chemokines with both readings in range (40%), followed by Bio-Rad® with magnetic beads (35%). Regarding reproducibility, the Millipore™ kit had the highest percentage (60%) of cytokines/chemokines with acceptable limits of agreement (<30%), followed by the Invitrogen™ with magnetic beads (40%) that had tighter limits of agreement.

Conclusions/Significance

Currently available kits for cytokine and chemokine quantification differ in reproducibility and concentration range of accurate detection. Luminex®-based kits with magnetic beads perform the best. Data highlights the importance of testing different kits before each study to choose the most appropriate, depending on the priority of the cytokines assessed.     

Comparison of Eleven Methods for Genomic DNA Extraction Suitable for Large-Scale Whole-Genome Genotyping and Long-Term DNA Banking Using Blood Samples

Over the recent years, next generation sequencing and microarray technologies have revolutionized scientific research with their applications to high-throughput analysis of biological systems. Isolation of high quantities of pure, intact, double stranded, highly concentrated, not contaminated genomic DNA is prerequisite for successful and reliable large scale genotyping analysis. High quantities of pure DNA are also required for the creation of DNA-banks. In the present study, eleven different DNA extraction procedures, including phenol-chloroform, silica and magnetic beads based extractions, were examined to ascertain their relative effectiveness for extracting DNA from ovine blood samples. The quality and quantity of the differentially extracted DNA was subsequently assessed by spectrophotometric measurements, Qubit measurements, real-time PCR amplifications and gel electrophoresis. Processing time, intensity of labor and cost for each method were also evaluated. Results revealed significant differences among the eleven procedures and only four of the methods yielded satisfactory outputs. These four methods, comprising three modified silica based commercial kits (Modified Blood, Modified Tissue, Modified Dx kits) and an in-house developed magnetic beads based protocol, were most appropriate for extracting high quality and quantity DNA suitable for large-scale microarray genotyping and also for long-term DNA storage as demonstrated by their successful application to 600 individuals.     

Infected foot ulcers in male and female diabetic patients: a clinico-bioinformative study

Background

Screening women for Chlamydia trachomatis infection in developing countries is highly desirable because of asymptomatic infection. The existing diagnostic methods in developing countries are not effective and their sensitivity fall below 45.0% which leads to further spread of infection. There is an urgent need for improved and cost effective diagnostic tests that will reduce the burden of sexually transmitted infections in the developing world.

Methods

Prevalence of C. trachomatis infection among women visiting gynaecology department of Hindu Rao hospital in Delhi, India was determined using Roche Amplicor Multi Well Plate kit (MWP) as well as using in-house PCR assay. We used 593 endocervical swabs for clinical evaluation of the in-house developed assay against Direct Fluorescence Assay (DFA; Group I n = 274) and Roche Amplicor MWP kit (Group II, n = 319 samples) and determined the sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV) of the in-house developed assay.

Results

We detected 23.0% positive cases and there was a higher representation of women aged 18-33 in this group. An in-house PCR assay was developed and evaluated by targeting unique sequence within the gyrA gene of C. trachomatis. Specificity of the reaction was confirmed by using genomic DNA of human and other STI related microorganisms as template. Assay is highly sensitive and can detect as low as 10 fg of C. trachomatis DNA. The resolved sensitivity of in-house PCR was 94.5% compared with 88.0% of DFA assay. The high specificity (98.4%) and sensitivity (97.1%) of the in-house assay against Roche kit and availability of test results within 3 hours allowed for immediate treatment and reduced the risk of potential onward transmission.

Conclusions

The in-house PCR method is cost effective (~ 20.0% of Roche assay) and hence could be a better alternative for routine diagnosis of genital infection by C. trachomatis to facilitate improved screening and treatment management.     

Analytical sensitivity and clinical performance of "COVID-19 RT-PCR Real TM FAST (CY5) (ATGen,Uruguay) and "ECUGEN SARS-CoV-2 RT-qPCR" (UDLA-STARNEWCORP,Ecuador)": High quality-low cost local SARS-CoV-2 tests for South America

BackgroundDozens of commercial RT-qPCR kits for SARS-CoV-2 detection are available with or without Emergency Use Authorization (EUA) by FDA or other regulatory agencies.ObjectiveWe evaluated the clinical performance of two SARS-CoV-2 RT-PCR kits designed and produced in South America, "COVID-19 RT-PCR Real TM FAST (CY5)" (ATGen, Uruguay) and "ECUGEN SARS-CoV-2 RT-qPCR" (UDLA-STARNEWCORP, Ecuador), for RT-qPCR SARS-CoV2 detection using "TaqMan 2019-nCoV Assay Kit v1" (Thermofisher, USA) as a gold standard technique.ResultsWe report a great clinical performance and analytical sensitivity for the two South American kits with sensitivity values of 96.4 and 100%, specificity of 100% and limit of detection in the range of 10 copies/uL of RNA extraction.Conclusions"COVID-19 RT-PCR Real TM FAST (CY5)" and "ECUGEN SARS-CoV-2 RT-qPCR" kits are reliable SARS-CoV-2 tests made in South America that have been extensively used in Uruguay, Argentina, Brazil, Bolivia and Ecuador. These locally produced SARS-CoV-2 tests have contributed to overcome supply shortages and reduce diagnosis cost, while maintaining the high quality standards of FDA EUA commercially available kits. This approach could be extended for other diagnostic products to improve infectious diseases surveillance at middle and low income countries beyond COVID-19 pandemic.     

Long COVID and its Management

The pandemic of COVID-19 is the biggest public health crisis in 21^st Century. Besides the acute symptoms after infection, patients and society are also being challenged by the long-term health complications associated with COVID-19, commonly known as long COVID. While health professionals work hard to find proper treatments, large amount of knowledge has been accumulated in recent years. In order to deal with long COVID efficiently, it is important for people to keep up with current progresses and take proactive actions on long COVID. For this purpose, this review will first introduce the general background of long COVID, and then discuss its risk factors, diagnostic indicators and management strategies. This review will serve as a useful resource for people to understand and prepare for long COVID that will be with us in the foreseeable future.     

Comparison of Manual and Automatic (MagNA Pure) Isolation Methods of Total RNA from Adipose Tissue

Aim Comparison of manual and automatic (MagNA Pure) isolation methods of total RNA from adipose tissue with respect to its quality and recovery factor. Material 120 human subcutaneous adipose tissue samples (about 100 mg/sample) were collected from patients during surgical operations. The tissue sample was stabilized in RNAlater (QIAGEN GmbH, Germany). Methods Total RNA was extracted by the following kits: Rneasy Protect Mini, Rneasy Lipid Tissue (QIAGEN GmbH, Germany) and MagNA Pure Compact RNA Isolation (Tissue) for MagNA Pure Compact Instrument (Roche Diagnostics GmbH, Germany). Results The average RNA yields with Rneasy Lipid Tissue kits were about two-fold higher in comparison with the Rneasy Protect Mini kit. When the MagNA Pure Compact System was used, RNA yields from the same sample were more uniform compared with manual systems. It was also more convenient and less time-consuming than the manual approach. No DNA contamination of total RNA samples was detected except for samples isolated by Rneasy Protect Mini Kit. Conclusion Rneasy Lipid Tissue Kit and MagNA Pure Compact RNA Isolation Kit (Tissue) provide RNA samples of high quantity, purity and PCR amplificability. RNA samples are suitable for further processing using methods of molecular biology.     

Adsorption of charged substrates and products on an enzyme reactor prepared by glutaraldehyde coupling on alkylamine derivatives of Ti(IV)-coated porous silica beads

Ti(IV) coating of porous silica beads, followed by derivatization with 1,6-diaminohexane and activation with glutaraldehyde was tested for the immobilization of glutamate decarboxylase (l-glutamate 1-carboxylyase, EC 4.1.1.15). The enzyme column prepared with the immobilized glutamate decarboxylase was designed for the preparation of 1 μmol γ-[¹³N]aminobutyric acid, a new tracer for positron emission tomography. Preliminary results, indicating high immobilization yields of active enzyme with good long term stabilities, led to a more detailed investigation of the Ti(IV) coating. When a column, containing about 1 g of enzyme-loaded beads was used for the synthesis of γ-[¹³N]aminobutyric acid (GABA) from l-[¹³N]glutamate, most of the¹³N activity remained adsorbed onto the column. The elution patterns of l-glutamate and GABA from columns of glutamate decarboxylase, immobilized on Ti(IV) coated silica beads, were investigated by using an h.p.l.c. u.v. detector. Different treatments of the Ti(IV) coated supports were tested to improve the desorption kinetics of GABA and l-glutamate. None of these methods gave a satisfactory improvement of the elution patterns of GABA and l-glutamate. The results indicate that the Ti(IV) coated silica beads have a large adsorption capacity, even though the enzyme is covalently linked. The described immobilization method is not recommended for enzymes having charged substrates or products and in which a small amount of substrate has to be applied onto a reactor containing a large amount of Ti(IV) coated support. The method can be applied when the enzyme reactor is operated in steady state conditions with continuous supply of substrate.     

Spermatozoa input concentrations and RNA isolation methods on RNA yield and quality in bull (Bos taurus)

Sperm RNA can be used to understand the past spermatogenic process, future successful fertilization, and embryo development. To study the sperm RNA composition and function, isolation of good quality RNA with sufficient quantity is essential. The objective of this study was to assess the influence of sperm input concentrations and RNA isolation methods on RNA yield and quality in bull sperm. The fresh semen samples from bulls (n = 6) were snap-frozen in liquid nitrogen and stored at −80 °C. The sperm RNA was isolated using membrane-based methods combined with TRIzol (RNeasy + TRIzol and PureLink + TRIzol) and conventional methods (TRIzol, Double TRIzol, and RNAzol RT). Based on fluorometric quantification, combined methods resulted in significantly (P < 0.05) higher total RNA yields (800–900 ng/30–40 × 10⁶) as compared with other methods and yielded 20 to 30 fg of RNA/spermatozoon. The quality of RNA isolated by membrane-based methods was superior to that isolated by conventional methods. The sperm RNA was observed to be intact as well as fragmented (50–2000 bp). The study revealed that the membrane-based methods with a cocktail of lysis solution and an optimal input concentration of 30 to 40 million sperm were optimal for maximum recovery of RNA from bull spermatozoa.