Hypermethylation and down-regulation of vitamin D receptor (VDR) as contributing factors for polycystic ovary syndrome (PCOS): a case-control study from Kashmir, North India.

BACKGROUND: Polycystic ovarian syndrome (PCOS) is a prevailing endocrinopathy affecting a significant population of women of reproductive age across the globe. A myriad set of complex intertwined factors ranging from etiological, genetic, and epigenetic reasons cause this disorder. Out of the different factors, vitamin D shows an imperative aspect in health and fertility of women with polycystic ovary syndrome (PCOS). The importance of vitamin D is facilitated by vitamin D receptor (VDR), a ligand-dependent transcription factor in the steroid/ thyroid hormone receptor superfamily that controls the pleiotropic biological properties of vitamin D. PURPOSE: The purpose of this study was to evaluate the role of promoter methylation of the VDR gene, a transcription factor with numerous biological utilities, with its relative expression and clinico-pathological findings and outcomes. METHODOLOGY: A total of 200 blood samples were collected, 100 from PCOS case subjects, and 100 from the normal healthy controls respectively, which were assessed by qRT-PCR for determining the expression summary. MS-PCR technique was used for analyzing the promoter methylation status of the VDR gene. Blood samples were withdrawn, respectively, for each case and the control study separately experimented for different stages for the given study, of which estimation of vitamin D was also a part. RESULTS: In this test-versus-control study, first, the promoter methylation status of VDR gene was identified which was found more prominent i.e., hyper-methylation of the VDR gene was identified in 84 cases (84%), and in the normal healthy controls, it was found (62%). The promoter methylation status of the VDR gene has remarkably shown the results with a significant difference (p value < 0.0001*). Second, the expression analysis of VDR gene was found to be strongly downregulated in majority (64%) of PCOS case samples analyzed by means fold change of 0.8743 (± 0.06466) (p value 0.0054**). This result is, therefore, indicative of VDR gene role in PCOS pathogenesis as the said gene is downregulated. Moreover, compared to the vitamin D parameter, hyper-methylation and expression analysis of the VDR promoter gene were found to correspond to some associations with PCOS. Certain case-and-control study analyses showed that patients with normal vitamin D levels showed less indicative effects of PCOS and vice versa. CONCLUSION: Our study, being exclusive from Kashmir, one of the foremost specified that VDR confirms anomalous methylation configuration in PCOS with subsequent downregulation in the gene expression i.e., there is an inverse correlation among VDR gene expression (downregulated) and methylation status (hyper-methylated) from the conclusion of our PCOS case-versus-control study.

Optimization of mRNA extraction from human nasal mucosa biopsies for gene expression profile analysis by qRT-PCR.

Quantitative real-time reverse transcriptase polymerase chain reaction (qRT-PCR) is the gold-standard method for analyzing modifications in gene expression in cells and tissues. However, large quantities of high-quality RNA samples are needed for analyzing the expression of multiple genes from one human tissue sample. Here, we provide an optimized protocol for extracting large amounts of RNA from human nasal mucosal biopsies. The quality and quantity of samples were sufficient for qRT-PCR analyses of the expressions of various genes, in duplicate. In contrast to other protocols, we optimized RNA isolation to increase the amount from nasal biopsy samples for analyses of multiple genes. In most previous publications, expressions of only one or a few genes, including housekeeping genes, were analyzed because the amount of biological material was small. We were able to improve our protocol with respect to the yield and quality of RNA. This is likely to produce better results from molecular analyses of very small biopsy samples of human nasal mucosa.

Comparison of Four Methods of RNA Extraction and cDNA Synthesis from The Venom of Peruvian Snakes of the Genus Bothrops of Clinical Importance.

RNA purification and cDNA synthesis represents the starting point for molecular analyses of snake venom proteins-enzymes. Usually, the sacrifice of snakes is necessary for venom gland extraction to identify protein-coding transcripts; however, the venom can be used as a source of transcripts. Although there are methods for obtaining RNA from venom, no comparative analysis has been conducted in the Bothrops genus. In the present study, we compared four commercial methods for RNA purification and cDNA synthesis from venom (liquid, lyophilized, or long-term storage) of four clinically relevant species of Peruvian Bothrops. Our results show that the TRIzol method presents the highest yield of RNA purified from venom (59 ± 11 ng/100 µL or 10 mg). The SuperScript First-Strand Synthesis System kit produced high amounts of cDNA (3.2 ± 1.2 ng cDNA/ng RNA), and the highest value was from combination with the Dynabeads mRNA DIRECT kit (4.8 ± 2.0 ng cDNA/ng RNA). The utility of cDNA was demonstrated with the amplification of six relevant toxins: thrombin-like enzymes, P-I and P-III metalloproteinases, acid and basic phospholipases A2, and disintegrins. To our knowledge, this is the first comparative study of RNA purification and cDNA synthesis methodologies from Bothrops genus venom.

MN/CA9 gene expression as a potential tumor marker for renal cell carcinoma.

MN/CA9 is a cell surface glycoprotein and a tumor-associated antigen. It plays a crucial role in the regulation of cell proliferation and oncogenesis. There is no ideal tumor marker currently available for renal cell carcinoma (RCC) with sufficient sensitivity and specificity. Therefore, we studied MN/CA9 gene expression in the tumor tissue, apparently normal kidney tissue, preoperative blood, and urine samples of patients with RCC. We included thirty cases of renal tumors (26 RCC and 4 benign tumors) in the study. We applied an RT-PCR assay for MN/CA9 gene expression to 26 RCC kidney tumor samples and four benign kidney tumor tissue samples. We also evaluated MN/CA9 gene expression in preoperative blood and urine samples of 15 of these cases. Additionally, thirty-five grossly normal renal tissue samples, including 21 from kidneys with RCC, were also evaluated for gene expression. The RT-PCR analysis revealed that twenty-one out of 26 RCC tissue samples showed MN/CA9 gene expression compared to three out of 35 non-malignant renal tissue samples (p < 0.05). Two out of four benign renal tissue samples also expressed this gene. We also observed MN/CA9 gene expression in nine out of 15 blood samples and four out of 15 urine samples. All patients with urinary MN/CA9 gene expression showed expression in blood and tumor tissue samples. We found a correlation in terms of MN/CA9 expression between blood and tumor tissue samples of RCC patients as those who exhibit MN/CA9 expression in blood were also positive at the tumor tissue levels. The difference in MN/CA9 gene expression in tumor tissue, blood, and urine samples in relation to the stage of the disease, nuclear grade, and histological cell-type was not statistically significant. However, all the three patients who had metastatic RCC had MN/CA9 gene expression in their blood. The existence of a tumor-associated antigen such as MN/CA9 may present a possible target for molecular diagnosis and management of RCC.

Thermostable properties of the equine infectious anemia virus nucleocapsid protein NCp11.

Retroviral nucleocapsid (NC) proteins are multifunctional nucleic acid binding proteins, playing critical roles in essentially every step of the viral replication cycle. As a small, basic protein, NC contains one or two highly conserved zinc-finger domains, each having an invariant CCHC motif, flanked by basic residues. In this study, we report for the first time, to our knowledge, the thermostable property of equine infectious anemia virus (EIAV) NCp11. About 43% of purified NCp11 remained soluble after incubation at 100 °C for 60 min, and heat-treated NCp11 maintained its abilities to bind to the E. coli RNA and the EIAV packaging signal sequence. At a very high degree of sequence occupancy, NCp11 inhibited first-strand cDNA synthesis catalyzed by either a commercial or the purified EIAV reverse transcriptase, and heat-treated NCp11 still inhibited the first-strand cDNA synthesis. We also found that protein concentrations, at a range from 0.1 to 0.9 µg/µl, have not affected the NCp11 thermostability significantly. However, NCp11 at acidic pH was more thermostable. Our findings highlight a new feature of the NC protein. Detailed understanding of NC's properties and functions will facilitate the development of effective and rational therapeutic strategies against retroviruses.

cDNA synthesis for BCR-ABL1 detection at the MMR level: the importance of using the appropriate kit.

BACKGROUND: The synthesis of complementary DNA (cDNA) for use in the detection of BCR-ABL1 at the Major Molecular Response (MMR) level is a well-established method used by clinical laboratories world-wide. However, the quality of cDNA provides sensitivity challenges and consequently affects the detection of Minimal Residual Disease (MRD). RESULTS: Herein, we evaluated six commercially available kits for the synthesis of cDNA according to amplification success rate, linearity and ABL1 copy number. Based on our results, the Invitrogen SuperScript® III Reverse Transcriptase kit performed better, among the ones used in this study, for the cDNA synthesis, followed by the First Strand cDNA Synthesis Kit for RT-PCR (AMV), available from Roche Applied Sciences. CONCLUSIONS: Accurate and sensitive testing for the detection of abnormal transcripts, allows the correct stratification and treatment of patients. Hence, the use of a suitable kit for the cDNA synthesis is of great importance. This study provides a comprehensive point of reference for clinical laboratories in an attempt to optimize BCR-ABL1 detection. We propose that the Invitrogen SuperScript® III Reverse Transcriptase kit is the most suitable, among the ones used in this study, for the cDNA synthesis to be used for the detection of BCR-ABL1 at the MMR level in a CML MRD assay.

Self-priming on the plant viral RNAs during reverse transcription-PCR.

The occurrence of the primer-independent cDNA synthesis during RT-PCR analysis of human and animal RNA viruses has been well documented. Conversely, there is scant knowledge about this event in plant RNA viruses. Here we show that the primer-independent cDNA synthesis occurs in all eight different plant RNA viruses tested in this study, suggesting a common phenomenon for RT-PCR analysis of plant RNA viruses. Additional experiments indicate that the event is likely contributed to by RNA self-priming, and can be effectively reduced or eliminated through increasing temperature of the RT reaction.

Applications of Quantitative PCR (qPCR) in Studies of Virus-Host Interactions.

Emerging viruses pose significant threats to human health and the global economy. In the past two decades, three different coronaviruses have emerged to cause worldwide public health concerns. The advent of high throughput genomic and transcriptomic technologies facilitated the study of virus-host interactions, accelerating the development of diagnostics, vaccines, and therapeutics. Here, we describe quantitative PCR (qPCR) in studies of virus-host interactions to dissect host responses and viral kinetics and how these relate to one another.

Genomic epidemiology of severe acute respiratory syndrome coronavirus 2 from Theni, Tamil Nadu.

Introduction: The coronavirus disease 2019 (COVID-19) is a viral infection characterized by respiratory and gastrointestinal symptoms. The causative agent of this infection is the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The genomic study helps in understanding the pathogenesis, epidemiology, and the development of therapeutic and preventive strategies in the combat against COVID-19. Materials and Methods: Nasopharyngeal and oropharyngeal swab samples were collected from asymptomatic and symptomatic patients during the time period of 2021-2022 for the detection of SARS-CoV-2 by employing real-time reverse transcriptase, cDNA synthesis, whole-genome sequencing by next-genome sequencing, analysis of SARS-CoV-2 sequence data and lineage and variant of concern assignment along with phylogenetic analysis. Results: Lineages BA.2.10 and BA.4.1.1 clustered with genomes from Senegal suggested the spread of infections. Similarly, high clustering among delta samples during the second wave showed possible importation and subsequent spread via local transmission. Conclusions: Studies like these are important to understand the characteristics and origins of locally circulating SARS-CoV-2 diversity in order to prevent further spread.

In vitro Isolation and Characterization of Multipotent Postnatal Stem Cells from Human Dental Pulp: An Approach for Regeneration of Neural Crest Tissue.

Postnatal dental pulp tissues give the proper justification of the stem cell assimilation and characteristic of the multipotent of the stem cells. Researchers use an in vitro isolation process for clarifying the different stages of staining and cell division. Data collected from various sources helps in understanding how the stem cells help in tissue regeneration. It highlights the immunological phenotypes with the synthesis with cDNA for mentioning molecular immunology. Study also mentions the mitochondrial consistency to measure the potentiality regarding the immunology and the way it differs from 0 to 21 days. Researchers also mention the way for the future development by utilizing the key advantages and definite multipotent of the dental stem cells.

Reverse Transcription Can Critically Impact the Diagnostic Outcome of BCR::ABL1 Quantitative Real-Time RT-PCR.

Reverse transcriptases (RT) are essential tools in BCR::ABL1 fusion transcript monitoring in chronic myeloid leukemia (CML). The RT type and cDNA priming method may impair the stoichiometry of cDNA synthesis, thereby potentially introducing a bias in BCR::ABL1 qRT-PCR data. Using the Acrometrix™ BCR::ABL1 reference panel and 37 clinical specimens, we have comparatively investigated the performance of the RTs MLV and SuperScript IV with random hexamer vs. target-specific priming. Quantitative RT-PCR results identified the priming type and RT type as major factors for diagnostic data variation, mainly due to the different efficacies of processing BCR::ABL1 low-copy-numbers (<50) compared to GUSB or ABL1 high-copy targets. The impairment of SuperScript IV in processing low- and high-copy-number RNA targets equally was not reflected by the diagnostically relevant Log (BCR::ABL1/GUSB%) values. Therefore, the correct representation of housekeeping and BCR::ABL1 target genes should have priority when aiming at as high a number of housekeeping gene copies as possible. Our data suggest that for improving BCR::ABL1 assay sensitivity, increased RNA/cDNA amounts and the use of distinct RT/priming combinations are advantageous. However, for inter-laboratory harmonization, the proper conversion factor according to the CML international standard (IS) has to be reevaluated each time the grade of RT is changed.

Use of a Novel Detection Tool to Survey Orthohantaviruses in Wild-Caught Rodent Populations.

Orthohantaviruses are negative-stranded RNA viruses with trisegmented genomes that can cause severe disease in humans and are carried by several host reservoirs throughout the world. Old World orthohantaviruses are primarily located throughout Europe and Asia, causing hemorrhagic fever with renal syndrome, and New World orthohantaviruses are found in North, Central, and South America, causing hantavirus cardiopulmonary syndrome (HCPS). In the United States, Sin Nombre orthohantavirus (SNV) is the primary cause of HCPS with a fatality rate of ~36%. The primary SNV host reservoir is thought to be the North American deer mouse, Peromyscus maniculatus. However, it has been shown that other species of Peromyscus can carry different orthohantaviruses. Few studies have systemically surveyed which orthohantaviruses may exist in wild-caught rodents or monitored spillover events into additional rodent reservoirs. A method for the rapid detection of orthohantaviruses is needed to screen large collections of rodent samples. Here, we report a pan-orthohantavirus, two-step reverse-transcription quantitative real-time PCR (RT-qPCR) tool designed to detect both Old and New World pathogenic orthohantavirus sequences of the S segment of the genome and validated them using plasmids and authentic viruses. We then performed a screening of wild-caught rodents and identified orthohantaviruses in lung tissue, and we confirmed the findings by Sanger sequencing. Furthermore, we identified new rodent reservoirs that have not been previously reported as orthohantavirus carriers. This novel tool can be used for the efficient and rapid detection of various orthohantaviruses, while uncovering potential new orthohantaviruses and host reservoirs that may otherwise go undetected.

Optimization of quantitative reverse transcription PCR method for analysis of weakly expressed genes in crops based on rapeseed.

Rapeseed (Brassica napus) is an allopolyploid hybrid (AACC genome) of turnip rape (B. rapa, genome: AA) and vegetable cabbage (B. oleraceae, genome: CC). Rapeseed oil is one of the main vegetable oils used worldwide for food and other technical purposes. Therefore, breeding companies worldwide are interested in developing rapeseed varieties with high yields and increased adaptation to harsh climatic conditions such as heat and prolonged drought. One approach to studying the mechanism of the epigenetically regulated stress response is to analyze the transcriptional changes it causes. In addition, comparing the expression of certain genes between stress- and non-stress-tolerant varieties will help guide breeding in the desired direction. Quantitative reverse transcription PCR (RT-qPCR) has been intensively used for gene expression analysis for several decades. However, the transfer of this method from model plants to crop species has several limitations due to the high accumulation of secondary metabolites, the higher water content in some tissues and therefore problems with their grinding and other factors. For allopolyploid rapeseed, the presence of two genomes, often with different levels of expression of homeologous genes, must also be considered. In this study, we describe the optimization of transcriptional RT-qPCR analysis of low-expression epigenetic genes in rapeseed, using Kinetochore Null2 (KNL2), a regulator of kinetochore complex assembly, as an example. We demonstrated that a combination of various factors, such as tissue homogenization and RNA extraction with TRIzol, synthesis of cDNA with gene-specific primers, and RT-qPCR in white plates, significantly increased the sensitivity of RT-qPCR for the detection of BnKNL2A and BnKNL2C gene expression.

Ready-to-use vertical flow paper device for instrument-free room temperature reverse transcription.

Paper-based nucleic acid detection and diagnosis are currently gaining much interest in point-of-care (POC) applications. The major steps involved in any nucleic acid amplification testing (NAAT) based diagnostics are nucleic acid isolation, reverse transcription (RT) (in the case of RNA), amplification and detection. RT is an important step in quantifying the viral load in case of disease diagnosis as well as quantifying gene expression levels in other molecular studies. cDNA synthesis is routinely carried out using a thermal cycler, with the process requiring temperatures between 40ºC to 65ºC. Here we report for the first time an instrument-free RT, performed at room temperature on cellulose-based paper devices. cDNA synthesis on paper was confirmed by RT-PCR and Sanger sequencing of the PCR products. Purified RNA from varied sources such as cell lysate, tissue and blood were used to test the methodology. Synthetic hepatitis C virus (HCV) RNA and human blood RNA were used as proof-of-concept to demonstrate the use of these devices in diagnostic applications. Further, ready-to-use paper-based reverse transcription (PRT) devices have been developed, wherein only the RNA sample is added on the device and the cDNA can be eluted after 30 min of incubation at room temperature. The devices were found to be stable for 30 days at - 20ºC storage. The cellulose-based PRT devices are simple, time saving and user-friendly for a complete instrument-free cDNA synthesis at room temperature.

Inactivation of Venezuelan Equine Encephalitis Virus Genome Using Two Methods.

Venezuelan equine encephalitis virus (VEEV) is an Alphavirus in the Togaviridae family of positive-strand RNA viruses. The viral genome of positive-strand RNA viruses is infectious, as it produces infectious virus upon introduction into a cell. VEEV is a select agent and samples containing viral RNA are subject to additional regulations due to their infectious nature. Therefore, RNA isolated from cells infected with BSL-3 select agent strains of VEEV or other positive-strand viruses must be inactivated before removal from high-containment laboratories. In this study, we tested the inactivation of the viral genome after RNA fragmentation or cDNA synthesis, using the Trinidad Donkey and TC-83 strains of VEEV. We successfully inactivated VEEV genomic RNA utilizing these two protocols. Our cDNA synthesis method also inactivated the genomic RNA of eastern and western equine encephalitis viruses (EEEV and WEEV). We also tested whether the purified VEEV genomic RNA can produce infectious virions in the absence of transfection. Our result showed the inability of the viral genome to cause infection without being transfected into the cells. Overall, this work introduces RNA fragmentation and cDNA synthesis as reliable methods for the inactivation of samples containing the genomes of positive-strand RNA viruses.

"Cells-to-cDNA on Chip": Phenotypic Assessment and Gene Expression Analysis from Live Cells in Nanoliter Volumes Using Droplet Microarrays.

In vitro cell-based experiments are particularly important in fundamental biological research. Microscopy-based readouts to identify cellular changes in response to various stimuli are a popular choice, but gene expression analysis is essential to delineate the underlying molecular dynamics in cells. However, cell-based experiments often suffer from interexperimental variation, especially while using different readout methods. Therefore, establishment of platforms that allow for cell screening, along with parallel investigations of morphological features, as well as gene expression levels, is crucial. The droplet microarray (DMA) platform enables cell screening in hundreds of nanoliter droplets. In this study, a "Cells-to-cDNA on Chip" method is developed enabling on-chip mRNA isolation from live cells and conversion to cDNA in individual droplets of 200 nL. This novel method works efficiently to obtain cDNA from different cell numbers, down to single cell per droplet. This is the first established miniaturized on-chip strategy that enables the entire course of cell screening, phenotypic microscopy-based assessments along with mRNA isolation and its conversion to cDNA for gene expression analysis by real-time PCR on an open DMA platform. The principle demonstrated in this study sets a beginning for myriad of possible applications to obtain detailed information about the molecular dynamics in cultured cells.

Profiling Long Non-coding RNA expression Using Custom-Designed Microarray.

Long non-coding RNAs (lncRNAs) are an important class of pervasive genes involved in a variety of biological functions. The abnormal expression of lncRNAs has been implicated in a range of many human diseases, including cancer. To date, a small number of functional lncRNAs have been well characterized. lncRNA expression profiling may help to identify useful molecular biomarkers and targets for novel therapeutic approaches. In this chapter, we describe a highly efficient lncRNA expression profiling method using a custom-designed microarray.

Quantification of long non-coding RNAs using qRT-PCR: comparison of different cDNA synthesis methods and RNA stability.

INTRODUCTION: Long non-coding RNAs (lncRNAs), a class of regulatory RNA molecules, are over 200 nucleotides long and could be used as a new potential biomarker, but their detection methods such as qRT-PCR are still not validated, and the influence of RNA degradation on lncRNA quantification is not clear. In this study, commercially available cDNA synthesis kits were tested and the influence of RNA degradation was compared. MATERIAL AND METHODS: Total RNA from FaDu cells was isolated and high quality RNA and highly degraded RNA samples were used. Reverse transcription was performed using three different commercially available kits and quantifications were performed using lncRNA Primer Plate and SYBR Green I Master by LightCycler 96. qRT-PCR was performed using three different cDNA samples and results are presented as the mean Ct values. A p-value < 0.05 was considered to be significant. RESULTS: Lower lncRNA Ct values (61/90; 67.78%) after qRT-PCR quantification were observed for cDNA synthesized using random hexamer primers preceded by polyA-tailing and adaptor-anchoring steps. It was observed that 9/90 (10.00%) lncRNAs were not detectable using different cDNA synthesis methods. For 75/90 (83%) lncRNAs, RNA degradation weakly influenced lncRNA Ct values and no differences were observed between high quality RNA and degraded samples. Seventy percent of examined lncRNAs showed significantly different Ct values depending on RNA degradation. CONCLUSIONS: cDNA synthesis kits with random hexamer primers preceded by polyA-tailing and adaptor-anchoring steps allows enhancement of lncRNA quantification specificity and sensitivity. In most cases degradation of RNA samples does not affect lncRNA quantification because these molecules have good stability.

Molecular Monitoring of Chronic Myeloid Leukemia.

Molecular diagnosis and measurement of minimal residual disease (MRD) in patients with chronic myeloid leukemia (CML) is essential for clinical management. In the era of tyrosine kinase inhibitor therapy molecular tests including BCR-ABL1 transcript monitoring and kinase domain mutation analysis are the main tools used to inform choice of treatment, appropriate dosage and even whether therapy can be safely withdrawn. Quantitation of BCR-ABL1 oncogene transcript by real-time quantitative PCR (qPCR) is currently the gold-standard method for monitoring as it provides superior sensitivity over karyotyping and fluorescent in situ hybridization (FISH). Here we describe step-by-step methods of RNA conversion to cDNA along with the qPCR protocol which is used in one of the main reference laboratories for this test.

The attachment of a DNA-binding Sso7d-like protein improves processivity and resistance to inhibitors of M-MuLV reverse transcriptase.

Reverse transcriptases (RTs) are a standard tool in both fundamental studies and diagnostics. RTs should possess elevated temperature optimum, high thermal stability, processivity and tolerance to contaminants. Here, we constructed a set of chimeric RTs, based on the combination of the Moloney murine leukaemia virus (M-MuLV) RT and either of two DNA-binding domains: the DNA-binding domain of the DNA ligase from Pyrococcus abyssi or the DNA-binding Sto7d protein from Sulfolobus tokodaii. The processivity and efficiency of cDNA synthesis of the chimeric RT with Sto7d at the C-end are increased several fold. The attachment of Sto7d enhances the tolerance of M-MuLV RT to the most common amplification inhibitors: NaCl, urea, guanidinium chloride, formamide, components of human whole blood and human blood plasma. Thus, fusing M-MuLV RT with an additional domain results in more robust and efficient RTs.