scholarly journals Identification of miR-29c-3p as a Robust Normalizer for Urine microRNA Studies in Bladder Cancer

Biomedicines ◽  
2020 ◽  
Vol 8 (11) ◽  
pp. 447
Author(s):  
Julia Oto ◽  
Emma Plana ◽  
Álvaro Fernández-Pardo ◽  
Fernando Cana ◽  
Manuel Martínez-Sarmiento ◽  
...  
Keyword(s):  
Bladder Cancer ◽  
Diagnostic Marker ◽  
Quantitative Polymerase Chain Reaction ◽  
Diagnostic Techniques ◽  
Expression Study ◽  
Non Invasive ◽  
Good Expression ◽  
Polymerase Chain ◽  
Early Diagnose ◽  
Independent Cohort

Bladder cancer (BC) is among the most frequent malignancies worldwide, being the most expensive cancer to treat and monitor and the most lethal urological cancer. Urine microRNAs (miRNAs) have been proposed as novel non-invasive biomarkers to early diagnose and monitor BC patients in order to avoid the performance of current aggressive diagnostic techniques. However, huge discrepancies arise among studies mainly due to the lack of standardization in the normalization, a crucial step in all miRNA studies. Our aim was to identify the best miRNA normalizer for miRNA studies in urine of BC patients. We evaluated the performance of 110 candidate miRNAs in urine of 35 BC patients and 15 healthy controls by Real Time quantitative Polymerase Chain Reaction (RT-qPCR) followed by a stability analysis with RefFinder. In this screening stage, miR-29c-3p arose as the most stably expressed miRNA in BC and controls, with a good expression level. Stability of miR-29c-3p expression was validated in an independent cohort of 153 BC patients and 57 controls. Finally, we evaluated the robustness of miR-29c-3p as normalizer in the expression study of miR-200c-3p, a potential diagnostic marker for BC. We propose miR-29c-3p as a normalizer for miRNA studies in BC urine. This is the first study that characterizes a reliable normalizer that may allow the comparison of future urine miRNA studies as non-invasive biomarkers for BC diagnosis and monitoring.

scholarly journals Identification of miR-20a-5p as Robust Normalizer for Urine microRNA Studies in Renal Cell Carcinoma and A Profile of Dysregulated microRNAs

2021 ◽  
Vol 22 (15) ◽  
pp. 7913
Author(s):  
Julia Oto ◽  
Raquel Herranz ◽  
Emma Plana ◽  
José Vicente Sánchez-González ◽  
Javier Pérez-Ardavín ◽  
...  
Keyword(s):  
Renal Cell Carcinoma ◽  
Cell Carcinoma ◽  
Renal Cell ◽  
Diagnostic Value ◽  
Diagnostic Model ◽  
Low Grade ◽  
Non Invasive ◽  
Good Expression ◽  
Independent Cohort

Renal cell carcinoma (RCC) is the third most frequent urinary malignancy and one of the most lethal. Current diagnostic and follow-up techniques are harmful and unspecific in low-grade tumors. Novel minimally invasive markers such as urine microRNAs (miRNAs) are under study. However, discrepancies arise among studies in part due to lack of consent regarding normalization. We aimed to identify the best miRNA normalizer for RCC studies performed in urine samples together with a miRNA profile with diagnostic value and another for follow-up. We evaluated the performance of 120 candidate miRNAs in the urine of 16 RCC patients and 16 healthy controls by RT-qPCR followed by a stability analysis with RefFinder. In this screening stage, miR-20a-5p arose as the most stably expressed miRNA in RCC and controls, with a good expression level. Its stability was validated in an independent cohort of 51 RCC patients and 32 controls. Using miR-20a-5p as normalizer, we adjusted and validated a diagnostic model for RCC with three miRNAs (miR-200a-3p, miR-34a-5p and miR-365a-3p) (AUC = 0.65; Confidence Interval 95% [0.51, 0.79], p = 0.043). let-7d-5p and miR-205-5p were also upregulated in patients compared to controls. Comparing RCC samples before surgery and fourteen weeks after, we identified let-7d-5p, miR-152-3p, miR-30c-5p, miR-362-3p and miR-30e-3p as potential follow-up profile for RCC. We identified validated targets of most miRNAs in the renal cell carcinoma pathway. This is the first study that identifies a robust normalizer for urine RCC miRNA studies, miR-20a-5p, which may allow the comparison of future studies among laboratories. Once confirmed in a larger independent cohort, the miRNAs profiles identified may improve the non-invasive diagnosis and follow-up of RCC.

scholarly journals Total Versus Free Placental Growth Factor Levels in the Pathogenesis of Preeclampsia

Hypertension ◽  
2020 ◽  
Vol 76 (3) ◽  
pp. 875-883
Author(s):  
Edouard Lecarpentier ◽  
Zsuzsanna K. Zsengellér ◽  
Saira Salahuddin ◽  
Ambart E. Covarrubias ◽  
Agnes Lo ◽  
...  
Keyword(s):  
Growth Factor ◽  
Ex Vivo ◽  
Quantitative Polymerase Chain Reaction ◽  
Placental Growth Factor ◽  
Protein Levels ◽  
Key Characteristics ◽  
Human Blood Samples ◽  
Polymerase Chain ◽  
Low Levels ◽  
Independent Cohort

Elevated circulating sFLT-1 (soluble fms-like tyrosine kinase) and low levels of its ligand, PlGF (placental growth factor), are key characteristics of preeclampsia. However, it is unclear if the low levels of plasma PlGF noted during preeclampsia are due to decreased placental production of PlGF or due to binding of PlGF by increased circulating sFLT-1. Here, we describe a biochemical procedure to dissociate PlGF-sFLT-1 complex ex vivo and when used in conjunction with an immunoassay platform, demonstrate a method to measure total and free PlGF in human blood samples. Using this method, we noted that plasma free PlGF levels were significantly lower in preeclampsia (N=22) than in nonhypertensive controls (N=24; mean, 314 versus 686 pg/mL, P <0.05), but total PlGF levels were not different (mean, 822 versus 800 pg/mL, P =0.49). In contrast, total sFLT-1 levels were significantly higher in preeclampsia than in nonhypertensive controls (mean, 16 957 versus 3029 pg/mL, P <0.01) and sFLT-1 levels correlated with bound PlGF levels (bound PlGF=total PlGF−free PlGF) in these samples ( r 2 =0.68). We confirmed these findings in an independent cohort of subjects (N=49). Furthermore, we did not detect any difference in PlGF mRNA by quantitative polymerase chain reaction or in PlGF protein expression by immunohistochemistry in preeclamptic placentas when compared with nonhypertensive controls. In contrast, sFLT-1 mRNA and protein levels were upregulated in placentas from women with preeclampsia. Taken together with prior studies, our results provide evidence that decrease in circulating PlGF noted during preeclampsia is largely mediated by excess circulating sFLT-1.

scholarly journals Efficient Diagnosis of Ratoon Stunting Disease of Sugarcane by Quantitative PCR on Pooled Leaf Sheath Biopsies

Plant Disease ◽  
2016 ◽  
Vol 100 (12) ◽  
pp. 2492-2498 ◽  
Author(s):  
Anthony J. Young ◽  
Asuka Kawamata ◽  
Mark A. Ensbey ◽  
Eleanore Lambley ◽  
Catherine J. Nock
Keyword(s):  
Phase Contrast ◽  
Quantitative Polymerase Chain Reaction ◽  
Xylem Sap ◽  
Diagnostic Techniques ◽  
Leaf Sheath ◽  
Conventional Pcr ◽  
Ratoon Stunting Disease ◽  
Contrast Microscopy ◽  
Plate Counts ◽  
Polymerase Chain

Ratoon stunting disease (RSD), caused by the bacterium Leifsonia xyli subsp. xyli, is arguably one of the most devastating diseases of sugarcane. Four diagnostic techniques were compared for 100 fields of sugarcane (Saccharum interspecific hybrids) of unknown infection status. These were quantitative polymerase chain reaction on pooled leaf sheath biopsies (LSB-qPCR), conventional PCR on the same templates (LSB-PCR), evaporative-binding enzyme immunoassay (EB-EIA) coupled with phase contrast microscopy (PCM) on expressed xylem sap from the same fields, and conventional PCR on the same xylem sap samples. LSB-qPCR and LSB-PCR detected the causal agent in 27 and 18 fields, respectively, whereas, from samples of expressed xylem sap from the same fields, conventional PCR identified 12 infections and EB-EIA/PCM detected L. xyli subsp. xyli in 3 fields. The sensitivities of qPCR and PCR were approximately 103 and 104 CFU ml−1, respectively, determined from plate counts of a dilution series. Tests were conducted on a further 139 LSB samples from across the Australian industry, with qPCR and PCR diagnosing RSD in 31 and 25 fields, respectively. Using qPCR and PCR on LSB samples, RSD was diagnosed in a range of cultivars throughout the year, and qPCR and PCR could detect L. xyli subsp. xyli in sugarcane ranging from 3 months to greater than 1 year old.

scholarly journals Altered expression of small leucine-rich proteoglycans (Decorin, Biglycan and Lumican): Plausible diagnostic marker in urothelial carcinoma of bladder

Tumor Biology ◽  
2017 ◽  
Vol 39 (5) ◽  
pp. 101042831769911 ◽  
Author(s):  
Sandeep Appunni ◽  
Vivek Anand ◽  
Madhuram Khandelwal ◽  
Amlesh Seth ◽  
Sandeep Mathur ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Bladder Cancer ◽  
Urothelial Carcinoma ◽  
Messenger Rna ◽  
Quantitative Polymerase Chain Reaction ◽  
Rna Expression ◽  
Chain Reaction ◽  
Polymerase Chain ◽  
Lower Expression ◽  
Urothelial Carcinoma Of Bladder

Small leucine-rich proteoglycans are components of extracellular matrix that regulates neoplastic transformation. Among small leucine rich proteoglycans, Decorin, Biglycan and Lumican are most commonly implicated markers, and their expression is well studied in various malignancies. In this novel study, we have collectively evaluated expression of these three molecules in urothelial carcinoma of bladder. Thirty patients of confirmed untreated bladder cancer, 30 healthy controls for blood and 30 controls for adjacent non-tumour tissue were enrolled. Blood was collected from all subjects and tumour/adjacent normal tissue was obtained from the patients. Circulatory levels were estimated by enzyme-linked immunosorbent assay, relative messenger RNA expression by quantitative polymerase chain reaction and protein expression by immunohistochemistry and western-blotting. Circulatory levels of Biglycan (p = 0.0038) and Lumican (p < 0.0001) were significantly elevated, and that of Decorin (p < 0.0001) was significantly reduced in patients as compared with controls. Protein expression by immunohistochemistry and western-blotting showed elevated expression of Lumican and Biglycan and lower expression of Decorin in urothelial carcinoma of bladder. Quantitative polymerase chain reaction for messenger RNA expression from tissue specimens revealed significantly higher expression of Biglycan (p = 0.0008) and Lumican (p = 0.01) and lower expression of Decorin (p < 0.0001) in urothelial carcinoma of bladder. Out of all molecules receiver operating characteristic curve showed that the 0.207 ng/ml cut-off of serum Lumican provided optimum sensitivity (90.0%) and specificity (90.0%). Significant alteration of matrix small leucine-rich proteoglycans in urothelial carcinoma of bladder was observed. Higher expression of Lumican in Bladder cancer patients with the cut-off value of highest optimum sensitivity and specificity shows its importance as a potential non-invasive marker for early detection of UBC following further validation in large patient cohort.

scholarly journals Diagnosis of Schistosoma Infection in Non-Human Animal Hosts: A Systematic Review and Meta-Analysis

2021 ◽  
Author(s):  
Song Liang ◽  
Keerati Ponpetch ◽  
Yi-Biao Zhou ◽  
Jia-Gang Guo ◽  
Berhanu Erko ◽  
...  
Keyword(s):  
Systematic Review ◽  
Meta Analysis ◽  
Quantitative Polymerase Chain Reaction ◽  
Diagnostic Techniques ◽  
Molecular Technique ◽  
Significant Heterogeneity ◽  
Animal Hosts ◽  
Polymerase Chain ◽  
Human Animal ◽  
Meta Analyses

Background Reliable and field-applicable diagnosis of schistosome infections in non-human animals is important for surveillance, control, and verification of interruption of human schistosomiasis transmission. This study aimed to summarize uses of available diagnostic techniques through a systematic review and meta-analysis. Methods and principal findings We systematically searched the literature and reports comparing two or more diagnostic tests in non-human animals for schistosome infection. Out of 4,909 articles and reports screened, 18 met our inclusion criteria, four of which were considered in the meta-analysis. A total of 14 techniques (parasitologic, immunologic, and molecular) and nine types of non-human animals were involved in the studies. Notably, four studies compared parasitologic tests (miracidium hatching test (MHT), Kato-Katz (KK), the Danish Bilharziasis Laboratory technique (DBL), and formalin-ethyl acetate sedimentation-digestion (FED-SD)) with quantitative polymerase chain reaction (qPCR), and sensitivity estimates (using qPCR as the reference) were extracted and included in the meta-analyses, showing significant heterogeneity across studies and animals hosts. The pooled estimate of sensitivity was 0.21 (95% confidence interval (CI): 0.03 &ndash; 0.48) with FED-SD showing highest sensitivity (0.89, 95% CI: 0.65 &ndash; 1.00). Conclusions and significance Our findings suggest that the parasitologic technique FEA-SD and the molecular technique, qPCR, are the most promising field-applicable techniques for schistosome diagnosis in non-human animal hosts. Future studies are needed for validation and standardization of the techniques for real-world field applications.

scholarly journals Urinary cell-free nucleic acid IQGAP3: a new non-invasive diagnostic marker for bladder cancer

Oncotarget ◽  
2018 ◽  
Vol 9 (18) ◽  
pp. 14354-14365 ◽  
Author(s):  
Won Tae Kim ◽  
Ye Hwan Kim ◽  
Pildu Jeong ◽  
Sung-Pil Seo ◽  
Ho-Won Kang ◽  
...  
Keyword(s):  
Bladder Cancer ◽  
Nucleic Acid ◽  
Diagnostic Marker ◽  
Non Invasive

scholarly journals Diagnostic accuracy of Fusobacterium nucleatum IgA and IgG ELISA test in colorectal cancer

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Melike Kurt ◽  
Zeki Yumuk
Keyword(s):  
Colorectal Cancer ◽  
Quantitative Polymerase Chain Reaction ◽  
Invasive Procedure ◽  
Fusobacterium Nucleatum ◽  
Elisa Test ◽  
Enzyme Linked Immunosorbent Assay ◽  
Igg Antibodies ◽  
Discriminative Ability ◽  
Non Invasive ◽  
Polymerase Chain

AbstractThe colorectal cancer is a serious health problem. The diagnosis of the disease mostly relies on an invasive procedure. A non-invasive diagnostic test such as an immunoassay, may facilitate diagnosis of colorectal cancer. The purpose of the study was to evaluate the use of antibodies against Fusobacterium nucleatum in the diagnosis of colorectal cancer (CRC). Totally 78 patients in three groups were included in the study. F. nucleatum in the tissues was detected using quantitative polymerase chain reaction assay. F. nucleatum IgA and IgG were measured using enzyme linked immunosorbent assay. F. nucleatum was detected in 86.7% and 73.1% cases of CRC and precancerous-benign colon disease (P-BCD), respectively. The OD values from F. nucleatum IgA and IgG ELISA tests were higher in CRC group compared with healthy individuals. The sensitivity of IgA ELISA test varied between 31.8 and 95.5% depending on the chosen cut-off values. The positivity rate of antibodies in patients with high amount of F. nucleatum in tissue was significantly greater than in the negative group. The F. nucleatum IgA and IgG antibodies in CRC were higher than the ones in healthy controls but the discriminative ability of the ELISA test was not adequate to be considered as a diagnostic tool.

scholarly journals Establishment of Optimal Housekeeping Genes for Urinary Extracellular Vesicle Biomarker Development: A Step Towards Non-Invasive Diagnostics

2021 ◽  
Author(s):  
Anula Divyash Singh ◽  
Rajeswari Koyyada ◽  
Rasmita Samal ◽  
Syed Baseeruddin Alvi ◽  
Sreekanth Patnam ◽  
...  
Keyword(s):  
Gene Expression ◽  
Quantitative Polymerase Chain Reaction ◽  
Housekeeping Genes ◽  
Extracellular Vesicle ◽  
Distribution Profile ◽  
Non Invasive ◽  
Expression Studies ◽  
Polymerase Chain ◽  
Gene Expression Studies ◽  
Invasive Diagnostics

Abstract Background. Urinary extracellular vesicles (UEVs) hold RNAs and can find their application in multiplex biomarker development. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) is the most commonly used method for gene expression studies. However, there are no reported optimal housekeeping genes available to normalize qPCR data for the UEVs RNA pool.Methods. UEVs were precipitated by Polyethylene glycol (P.E.G. Mn6000) from the urine of 40 human individuals and characterized for their molecular, biophysical, and biochemical purity and integrity. In addition, the expression of five commonly used housekeeping genes B2M, RPL13A, PPIA, HMBS, and GAPDH, were quantified by qPCR in the UEV RNA pool and analyzed through algorithms of NormFinder, geNorm, BestKeeper, and Delta Ct integrated into ReFinder.Results: 12% PEG precipitation yielded round and cup-shaped UEVs. The size and distribution profile of UEVs were around 30 – 100nm through electron microscopy, NTA, and DLS. Acetylcholine esterase and Dipeptidyl peptidase-IV activity were used to arrive at their functional purity. B2M and RPL13A genes were identified as stable genes with a mean stability score of 1.5(geNorm) and below 1 (NormFinder) through ReFinder, which yield comprehensive ranking analysisConclusions: B2M and RPL13A are optimal reference genes and can be used for UEVs based gene expression studies.

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