scholarly journals Degradation of serum microRNAs during transient storage of serum samples at 4℃

2017 ◽  
Vol 55 (1) ◽  
pp. 178-180 ◽  
Author(s):  
Toshiko Aiso ◽  
Shu Takigami ◽  
Akiko Yamaki ◽  
Hiroaki Ohnishi
Keyword(s):  
Extracellular Vesicles ◽  
Room Temperature ◽  
The Other ◽  
Transient Storage ◽  
Plasma Samples ◽  
Serum Samples ◽  
Prolonged Incubation ◽  
Circulating Micrornas ◽  
Non Invasive ◽  
Serum Micrornas

Background Numerous studies demonstrate the potential of circulating microRNAs as non-invasive biomarkers for several diseases. Circulating microRNAs are much more stable than mRNAs and remain largely intact even after prolonged incubation at room temperature. However, recent reports show that microRNAs in serum or plasma samples have diverse stabilities. The aim of this pilot study is to evaluate the stabilities of miR-92a, miR-122 and miR-145 in serum during transient storage at 4℃ before freezing. Methods Serum samples were stored for 24 h at 4℃, and then RNA was extracted from whole serum or extracellular vesicles in serum. Total Exosome Isolation Reagent (from serum) was used for the fractionation of extracellular vesicles. Reverse transcription and real-time PCR of microRNAs were performed using the TaqMan MicroRNA Assays for miR-92a, miR-122 and miR-145. Results MiR-122 and miR-145 were degraded rapidly in serum; the concentrations dropped to 35.9% ( P < 0.001) and 29.3% ( P < 0.0001), respectively. These microRNAs in extracellular vesicles exhibited similar instability; the concentrations were 52.2% ( P < 0.05) and 56.5% ( P < 0.01), respectively. On the other hand, no significant degradation of miR-92a was observed (whole serum: P = 0.052, extracellular vesicles: P = 0.196). Conclusions MiR-122 and miR-145 in serum are extremely unstable and could be degraded during transient storage of serum at 4℃ prior to freezing.

scholarly journals IL8 and IL16 levels indicate serum and plasma quality

2018 ◽  
Vol 56 (7) ◽  
pp. 1054-1062 ◽  
Author(s):  
Olga Kofanova ◽  
Estelle Henry ◽  
Rocio Aguilar Quesada ◽  
Alexandre Bulla ◽  
Hector Navarro Linares ◽  
...  
Keyword(s):  
Diagnostic Performance ◽  
Room Temperature ◽  
Plasma Samples ◽  
Sample Processing ◽  
Serum Samples ◽  
Sample Quality ◽  
Edta Plasma ◽  
Citrate Plasma

Abstract Background: Longer pre-centrifugation times alter the quality of serum and plasma samples. Markers for such delays in sample processing and hence for the sample quality, have been identified. Methods: Twenty cytokines in serum, EDTA plasma and citrate plasma samples were screened for changes in concentration induced by extended blood pre-centrifugation delays at room temperature. The two cytokines that showed the largest changes were further validated for their “diagnostic performance” in identifying serum or plasma samples with extended pre-centrifugation times. Results: In this study, using R&D Systems ELISA kits, EDTA plasma samples and serum samples with a pre-centrifugation delay longer than 24 h had an IL16 concentration higher than 313 pg/mL, and an IL8 concentration higher than 125 pg/mL, respectively. EDTA plasma samples with a pre-centrifugation delay longer than 48 h had an IL16 concentration higher than 897 pg/mL, citrate plasma samples had an IL8 concentration higher than 21.5 pg/mL and serum samples had an IL8 concentration higher than 528 pg/mL. Conclusions: These robust and accurate tools, based on simple and commercially available ELISA assays can greatly facilitate qualification of serum and plasma legacy collections with undocumented pre-analytics.

MicroRNA profiling in serum: Potential signatures for breast cancer diagnosis

2020 ◽  
pp. 1-13
Author(s):  
Xuan Zou ◽  
Tiansong Xia ◽  
Minghui Li ◽  
Tongshan Wang ◽  
Ping Liu ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Mirna Expression ◽  
Expression Patterns ◽  
External Validation ◽  
Breast Cancer Diagnosis ◽  
Serum Samples ◽  
Circulating Micrornas ◽  
Non Invasive ◽  
Tumor Tissues ◽  
Serum Exosomes

BACKGROUND: Circulating microRNAs (miRNAs) prove to be potential non-invasive indicators of cancers. The purpose of this study is to profile serum miRNA expression in breast cancer (BC) patients to find potential biomarkers for BC diagnosis. METHODS: The miRNA expression patterns of serum samples from 216 BC patients and 214 normal control subjects were compared. A four-phase validation was conducted for biomarker identification. In the screening phase, the Exiqon miRNA qPCR panel was employed to select candidates, which were further analyzed by quantitative reverse transcriptase PCR in the following training, testing, and external validation phases. RESULTS: A 12-miRNA (let-7b-5p, miR-106a-5p, miR-19a-3p, miR-19b-3p, miR-20a-5p, miR-223-3p, miR-25-3p, miR-425-5p, miR-451a, miR-92a-3p, miR-93-5p, and miR-16-5p) panel in serum was constructed. The diagnostic performance of the panel was assessed using ROC curve analyses. The area under the curves (AUCs) were 0.952, 0.956, 0.941 and 0.950 for the four separate phases, respectively. Additionally, the expression features of the 12 miRNAs were further explored in 32 pairs of BC tumor and para-tumor tissues, and 32 pairs of serum exosomes samples from patients and healthy subjects. miR-16-5p, miR-106a-5p, miR-25-3p, miR-425-5p, and miR-93-5p were highly overexpressed and let-7b-5p was conversely downregulated in tumor tissues. Excluding miR-20a-5p and miR-223-3p, the 10 other miRNAs were all significantly upregulated in BC serum-derived exosomes. CONCLUSION: A signature consisting of 12 serum miRNAs was identified and showed potential for use in non-invasive diagnosis of BC.

Two-site time-resolved immunofluorometric assay of human insulin.

1986 ◽  
Vol 32 (4) ◽  
pp. 637-640 ◽  
Author(s):  
E Toivonen ◽  
I Hemmilä ◽  
J Marniemi ◽  
P N Jørgensen ◽  
J Zeuthen ◽  
...  
Keyword(s):  
Human Insulin ◽  
Fluorescence Enhancement ◽  
Microtiter Plate ◽  
The Other ◽  
Plasma Samples ◽  
Serum Samples ◽  
Time Resolved ◽  
Standard Curve ◽  
Sandwich Type ◽  
Plate Strip

Abstract We describe a two-site "sandwich"-type time-resolved immunofluorometric assay for human insulin, based on use of two monoclonal antibodies with different specificities. The first antibody is immobilized on the surface of microtiter plate strip wells, the other is labeled with Eu3+. Serum samples can be assayed with one incubation step; two incubation steps are required when plasma samples are assayed. After the immunoreactions are complete, the bound fraction of Eu3+-label is quantified by dissociating it in a fluorescence-enhancement solution and measuring its fluorescence with a fluorometer with time-resolution. The sensitivity of the assay is 0.24 micro-int. units/mL. The standard curve is linear from 0.24 to 2400 micro-int. units/mL.

scholarly journals Serum microRNAs are non-invasive biomarkers for the presence and progression of subarachnoid haemorrhage

2017 ◽  
Vol 37 (1) ◽  
Author(s):  
Nian-sheng Lai ◽  
Jia-qi Zhang ◽  
Fei-yun Qin ◽  
Bin Sheng ◽  
Xing-gen Fang ◽  
...  
Keyword(s):  
Subarachnoid Haemorrhage ◽  
Quantitative Pcr ◽  
Operating Characteristic ◽  
Roc Curves ◽  
Pcr Analysis ◽  
Healthy Controls ◽  
Modified Rankin Scale ◽  
Serum Samples ◽  
Non Invasive ◽  
Serum Micrornas

miRNAs are important regulators of translation and have been associated with the pathogenesis of a number of cardiovascular diseases including stroke and may be possible prognostic biomarkers. The purpose of the present study was to determine the expression levels of miRNAs in the sera of subarachnoid haemorrhage (SAH) patients and to evaluate their relationships with the severity and clinical outcome of SAH. Serum samples on day 3 after the onset of SAH were subjected to microarray analysis with Exqion miRCURYTM LNA array and quantitative PCR analysis. Serum samples from SAH patients (n=60) and healthy controls (n=10) were subjected to quantitative PCR analysis. The severities and clinical outcomes of the SAH patients were evaluated with the WFNS grade and the Modified Rankin Scale (mRS). Three miRNAs, miR-502-5p, miR-1297 and miR-4320 were significantly up-regulated in the sera of SAH patients when compared with the healthy controls. The serum miR-502-5p and miR-1297 levels were significantly higher in the patients with severe SAH and a poor outcome than in those with mild SAH and a good outcome (P<0.05). The areas under the receiver operating characteristic (ROC) curves (AUCs) of miR-502-5p, miR-1297 and miR-4320 to distinguish the SAH patients from the healthy controls were 0.958 (P<0.001), 0.950 (P<0.001) and 0.843 (P<0.001) respectively. Taken together, these results indicate that miR-502-5p and miR-1297 are potentially valuable indicators of the diagnosis, severity and prognosis of SAH, and miR-4320 was a potentially valuable indicator of the diagnosis of SAH.

scholarly journals Investigating the Use of Protein Saver Cards for Storage and Subsequent Detection of Bovine Anti-Brucella abortus Smooth Lipopolysaccharide Antibodies and Gamma Interferon

2013 ◽  
Vol 20 (11) ◽  
pp. 1669-1674 ◽  
Author(s):  
Lucy Duncombe ◽  
Nicola J. Commander ◽  
Sevil Erdenlig ◽  
John A. McGiven ◽  
Judy Stack
Keyword(s):  
Blood Serum ◽  
Brucella Abortus ◽  
Room Temperature ◽  
Plasma Samples ◽  
Bovine Blood ◽  
Bovine Brucellosis ◽  
Serum Samples ◽  
Content Type ◽  
Antibody Titers ◽  
Ifn Γ

ABSTRACTBrucella abortus, a smooth strain of the genusBrucella, is the causative agent of bovine brucellosis. To support the ongoing development of diagnostic tests for bovine brucellosis, the use of Protein Saver cards (Whatman) for bovine blood serum and plasma sample collection has been evaluated. These cards offer significant logistical and safety alternatives to transporting and storing liquid samples and may aid in diagnostic programs and validation studies. To evaluate the utility of these cards, 204 bovine blood serum samples fromBrucella-infected and noninfected animals were stored on and eluted from the Protein Saver cards. Anti-Brucellasmooth lipopolysaccharide (sLPS) antibody titers for the serum eluates were compared to those of the unprocessed original serum samples by indirect enzyme-linked immunosorbent assay (ELISA). The results showed a highly significant correlation between titers from the serum eluates and the unprocessed sera. Therefore, under these circumstances, serum eluates and unprocessed serum samples may be used interchangeably. Blood plasma from 113 mitogen-stimulated whole-blood samples was added to and eluted from the Protein Saver cards. The gamma interferon (IFN-γ) titers in the plasma eluates were compared to those of the unprocessed plasma samples obtained by IFN-γ ELISA. The results showed a significant correlation between the plasma eluates and the unprocessed plasma samples. To derive a signal in the plasma eluate, it was necessary to develop a novel and highly sensitive ELISA for the detection of IFN-γ. The serum samples stored on cards at room temperature over a 10-day period showed little variation in antibody titers. However, the plasma eluates showed a progressive loss of IFN-γ recovery over 10 days when stored at room temperature.

Measurement of serum and plasma osmolality in healthy young humans – influence of time and storage conditions

2004 ◽  
Vol 42 (8) ◽  
Author(s):  
Christian C. Seifarth ◽  
Johannes Miertschischk ◽  
Eckhardt G. Hahn ◽  
Johannes Hensen
Keyword(s):  
Room Temperature ◽  
Freezing Point ◽  
Plasma Osmolality ◽  
Storage Conditions ◽  
Plasma Samples ◽  
Freezing And Thawing ◽  
Serum Samples ◽  
Slow Increase ◽  
Stabilizing Agent ◽  
Different Temperatures

AbstractBackground: The precise measurement of osmolality is crucial in the differential diagnosis of disorders of water balance. Storage conditions, and freezing and thawing of serum or plasma samples before osmometry may influence the accuracy of measured values. Methods: A series of serum and plasma samples of 25 healthy young individuals were stored under different conditions at different temperatures (room temperature (22°C), 7°C, –21°C, –78°C) for up to 56 days. Before freezing a protein-stabilizing agent (bacitracin) was added to one part of the samples. Osmolality was examined using the freezing point method. Results: At room temperature osmolality was stable for up to 3 days but showed a tendency toward an increase that was significant on day 14. In contrast, at 7°C an initial significant decrease in serum osmolality occurred (day 1), which was followed by a slow increase. Serum samples stored at –21°C showed a significantly lower osmolality on the 14th day compared to baseline. Adding bacitracin before freezing reduced this decrease by more than half, but the deviation was still significant. In samples stored at –78°C no significant alteration of osmolality from baseline was observed over the observation period of 56 days if samples were thawed in a 37°C water bath. Conclusion: Immediate measurement of osmolality is most reliable in order to obtain accurate values, although storing at room temperature does not influence osmolality significantly during the first 3 days. If storage is necessary for longer, samples should be stored at –78°C and must be thawed quickly (at 37°C). Under these conditions reliable values can be obtained from frozen serum or plasma. Storage at 7°C is not recommended. If samples are stored at –21°C the addition of a protein-stabilizing agent may be useful.

Diagnostic value of oncofetal miRNAs in cancers: A comprehensive analysis of circulating miRNAs in pan-cancers and UCB

2021 ◽  
pp. 1-18
Author(s):  
Xin Zhou ◽  
Cheng Liu ◽  
Yin Yin ◽  
Cheng Zhang ◽  
Xuan Zou ◽  
...  
Keyword(s):  
Expression Patterns ◽  
Diagnostic Value ◽  
Comprehensive Analysis ◽  
Detection Methods ◽  
Plasma Samples ◽  
Circulating Mirnas ◽  
Serum Samples ◽  
Chinese Populations ◽  
Non Invasive ◽  
Plasma Mirnas

BACKGROUND: Circulating miRNAs are promising biomarkers for detection of various cancers. As a “developmental” disorder, cancer showed great similarities with embryos. OBJECTIVE: A comprehensive analysis of circulating miRNAs in umbilical cord blood (UCB) and pan-cancers was conducted to identify circulating miRNAs with potential for cancer detection. METHODS: A total of 3831 cancer samples (2050 serum samples from 15 types of cancers and 1781 plasma samples from 13 types of cancers) and 248 UCB samples (120 serum and 128 plasma samples) with corresponding NCs from Chinese populations were analyzed via consistent experiment workflow with Exiqon panel followed by multiple-stage validation with qRT-PCR. RESULTS: Thirty-four serum and 32 plasma miRNAs were dysregulated in at least one type of cancer. Eighteen serum and 16 plasma miRNAs were related with embryos. Among them, 9 serum and 8 plasma miRNAs with consistent expression patterns between pan-cancers and UCB were identified as circulating oncofetal miRNAs. Retrospective analysis confirmed the diagnostic ability of circulating oncofetal miRNAs for specific cancers. And the oncofetal miRNAs were mainly up-regulated in tissues of pan-cancers. CONCLUSIONS: Our study might serve as bases for the potential application of the non-invasive biomarkers in the future clinical.

scholarly journals Experimental Validation of a Potential Circulating microRNA Panel for Non-Invasive Pancreatic Cancer Diagnosis

2021 ◽  
Author(s):  
Ali Seyed Salehi ◽  
Negar Parsa ◽  
Farnaz Roshan-Farzad ◽  
Ali Behmanesh ◽  
Mohadeseh Fathi ◽  
...  
Keyword(s):  
Pancreatic Cancer ◽  
Differential Expression Analysis ◽  
External Validation ◽  
Diagnostic Value ◽  
Ductal Adenocarcinoma ◽  
Circulating Microrna ◽  
Plasma Samples ◽  
Serum Samples ◽  
Non Invasive ◽  
Testing Stage

Abstract MicroRNA (miRNA) expression dysregulations in pancreatic ductal adenocarcinoma (PDAC) have been studied widely for their diagnostic and prognostic utility. By the use of Bioinformatics-based methods, in our previous study, we identified some potential miRNA panels for diagnosis of Pancreatic cancer patients from non-cancerous controls (the screening stage). In this report, we used 142 plasma samples from people with and without pancreatic cancer (PC) to conduct RT-qPCR differential expression analysis to assess the strength of the first previously proposed diagnostic panel (consisting of miR-125a-3p, miR-4530 and miR-92a-2-5p). For this aim the research was divided into two phases: testing, and external validation. A total of 92 PC plasma samples and normal controls (NCs) were evaluated in the testing stage to determine the ability of the considered miRNAs to discriminate cancer from non-cancer samples as a panel. Furthermore, in the external validation phase, a group of 25 PC serum samples vs. 25 NCs was employed to validate the diagnostic value of the panel. As the result, we identified significant up-regulation for all the three considered miRNAs in the serum of PC patients. After that, a three-miRNA panel in serum was developed. For the testing, validating and combined stages, the area under the receiver operating characteristic curves (AUC) for the panel were 0.850, 0.910 and 0.86 respectively, indicating that it had a higher diagnostic value than individual miRNAs. Therefore, we detected a promising three-miRNA panel in the plasma for non-invasive PC diagnosis (miR-125a-3p, miR-4530 and miR-92a-2-5p).

Six osteocalcin assays compared

1994 ◽  
Vol 40 (11) ◽  
pp. 2071-2077 ◽  
Author(s):  
E M Díaz Diego ◽  
R Guerrero ◽  
C de la Piedra
Keyword(s):  
Amino Acids ◽  
Impaired Renal Function ◽  
Room Temperature ◽  
The Other ◽  
Serum Samples ◽  
Antibody Treatment ◽  
Intact Osteocalcin ◽  
Paget Disease Of Bone ◽  
Paget Disease ◽  
Human Osteocalcin

Abstract We evaluated comparatively six commercially available osteocalcin kits: ELSA-OST-NAT IRMA (CIS), ELSA-Osteo IRMA (CIS), Osteocalcin IRMA (Nichols), OSTK-PR RIA (CIS), OSCA Test Osteocalcin RIA (Henning), and Osteocalcin RIA (Nichols). All of them presented acceptable values of variance, sensitivity, and serial dilutions. However, only the ELSA-OST-NAT, ELSA-Osteo, and OSCA Test kits had analytical recoveries near 100% for added purified human osteocalcin; the other assays showed important differences in standardization. After treating samples with antibody against amino acids 43-49 of human osteocalcin, only the ELSA-OST-NAT IRMA detected no residual osteocalcin. Stability of serum values at room temperature and at 4 degrees C is good in the ELSA-Osteo and Osteocalcin IRMAs and in the Henning RIA if serum samples are collected in special tubes available with the kit. Differences in stability and after antibody treatment can be due to the degradation of intact osteocalcin molecules in serum and to osteocalcin fragments detected by assays. Results by different assays were well correlated for samples from controls and postmenopausal osteoporotic women but not for patients with impaired renal function or Paget disease of bone, probably because of osteocalcin fragments accumulated in serum of these patients and differences in specificity of the assays.

scholarly journals High-Throughput Sequencing of Circulating MicroRNAs in Plasma and Serum during Pregnancy Progression

Life ◽  
2021 ◽  
Vol 11 (10) ◽  
pp. 1055
Author(s):  
Elena S. Vashukova ◽  
Polina Y. Kozyulina ◽  
Roman A. Illarionov ◽  
Natalya O. Yurkina ◽  
Olga V. Pachuliia ◽  
...  
Keyword(s):  
High Throughput ◽  
Molecular Mechanisms ◽  
High Throughput Sequencing ◽  
Maternal Blood ◽  
P Value ◽  
Sequencing Technology ◽  
Serum Samples ◽  
Circulating Micrornas ◽  
Non Invasive ◽  
Qrt Pcr

Although circulating microRNAs (miRNAs) in maternal blood may play an important role in regulation of pregnancy progression and serve as non-invasive biomarkers for different gestation complications, little is known about their profile in blood during normally developing pregnancy. In this study we evaluated the miRNA profiles in paired plasma and serum samples from pregnant women without health or gestational abnormalities at three time points using high-throughput sequencing technology. Sequencing revealed that the percentage of miRNA reads in plasma and serum decreased by a third compared to first and second trimesters. We found two miRNAs in plasma (hsa-miR-7853-5p and hsa-miR-200c-3p) and 10 miRNAs in serum (hsa-miR-203a-5p, hsa-miR-495-3p, hsa-miR-4435, hsa-miR-340-5p, hsa-miR-4417, hsa-miR-1266-5p, hsa-miR-4494, hsa-miR-134-3p, hsa-miR-5008-5p, and hsa-miR-6756-5p), that exhibit level changes during pregnancy (p-value adjusted < 0.05). In addition, we observed differences for 36 miRNAs between plasma and serum (p-value adjusted < 0.05), which should be taken into consideration when comparing the results between studies performed using different biosample types. The results were verified by analysis of three miRNAs using qRT-PCR (p < 0.05). The present study confirms that the circulating miRNA profile in blood changes during gestation. Our results set the basis for further investigation of molecular mechanisms, involved in regulation of pregnancy, and the search for biomarkers of gestation abnormalities.

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