RNA isolation from human serum and plasma samples using the Norgen exosomal RNA purification mini kit

2015 ◽  
Author(s):  
Roger P. Alexander ◽  
Louise C. Laurent ◽  
Roger P. Alexander
Keyword(s):  
Human Serum ◽  
Rna Isolation ◽  
Plasma Samples ◽  
Rna Purification

scholarly journals Dihydro-sphingosine 1-phosphate interacts with carrier proteins in a manner distinct from that of sphingosine 1-phosphate

2018 ◽  
Vol 38 (5) ◽  
Author(s):  
Yuko Mishima ◽  
Makoto Kurano ◽  
Tamaki Kobayashi ◽  
Masako Nishikawa ◽  
Ryunosuke Ohkawa ◽  
...  
Keyword(s):  
Human Serum ◽  
Clinical Significance ◽  
Knockout Mice ◽  
Plasma Samples ◽  
Carrier Proteins ◽  
Serum Samples ◽  
Apolipoprotein M ◽  
Physiological Properties ◽  
Specific Manner ◽  
Sphingosine 1 Phosphate

Dihydro-sphingosine 1-phosphate (DH-S1P) is an analog of sphingosine 1-phosphate (S1P), which is a potent lysophospholipid mediator. DH-S1P has been proposed to exert physiological properties similar to S1P. Although S1P is known to be carried on HDL via apolipoprotein M (apoM), the association between DH-S1P and HDL/apoM has not been fully elucidated. Therefore, in the present study, we aimed to elucidate this association and to compare it with that of S1P and HDL/apoM. First, we investigated the distributions of S1P and DH-S1P among lipoproteins and lipoprotein-depleted fractions in human serum and plasma samples and observed that both S1P and DH-S1P were detected on HDL; furthermore, elevated amounts of DH-S1P in serum samples were distributed to the lipoprotein-depleted fraction to a greater degree than to the HDL fraction. Concordantly, a preference for HDL over albumin was only observed for S1P, and not for DH-S1P, when the molecules were secreted from platelets. Regarding the association with HDL, although both S1P and DH-S1P prefer to bind to HDL, HDL preferentially accepts S1P over DH-S1P. For the association with apoM, S1P was not detected on HDL obtained from apoM knockout mice, while DH-S1P was detected. Moreover, apoM retarded the degradation of S1P, but not of DH-S1P. These results suggest that S1P binds to HDL via apoM, while DH-S1P binds to HDL in a non-specific manner. Thus, DH-S1P is not a mere analog of S1P and might possess unique clinical significance.

scholarly journals Detection of Chromosome 21-encoded mRNA of Placental Origin in Maternal Plasma

2003 ◽  
Vol 49 (9) ◽  
pp. 1445-1449 ◽  
Author(s):  
Cees B M Oudejans ◽  
Attie T J J Go ◽  
Allerdien Visser ◽  
Monique A M Mulders ◽  
Bart A Westerman ◽  
...  
Keyword(s):  
Down Syndrome ◽  
Placental Tissue ◽  
Rna Isolation ◽  
First Trimester ◽  
Maternal Plasma ◽  
Chromosome 21 ◽  
Placental Lactogen ◽  
Plasma Samples ◽  
Human Placental Lactogen ◽  
Dnase Treatment

Abstract Background: mRNA of placental origin (i.e., human placental lactogen and β-human chorionic gonadotropin) has been demonstrated to be easily detectable in maternal plasma. We tested whether detection of chromosome 21-encoded mRNA of placental origin is possible in maternal plasma obtained during the first trimester. Methods: Plasma samples were obtained from pregnant women between weeks 9–13 of pregnancy. RNA was isolated from 800 or 1600 μL of plasma by silica-based affinity isolation and, after on-column DNase treatment, was subjected to two-step, one-tube reverse transcription-PCR with gene specific primers. Results: Three chromosome 21-encoded genes located within the Down syndrome critical region with overexpression in trisomy 21 placentas were screened for expression in early placental tissue to select their potential use for RNA based plasma screening. One of the chromosome 21-encoded genes (LOC90625) showed strong expression in first trimester placenta similar to CSH1 (human placental lactogen) and was selected for plasma analysis. The RNA isolation assay was validated with CSH1 mRNA, which could be detected in the plasma of all women tested in weeks 9–13 of pregnancy. RNA from the chromosome 21-encoded, placentally expressed gene, LOC90625, was present in maternal first-trimester plasma and could be detected in 60% of maternal plasma samples when 800 μL of plasma was used and in 100% of samples when 1600 μL of plasma was used. Conclusion: The detection of chromosome 21-encoded mRNA of placental origin in maternal plasma during the first trimester may allow development of plasma-RNA-based strategies for prenatal prediction of Down syndrome. LOC90625 is a candidate gene for this purpose.

LC–MS/MS measurement of 26 steroid hormones in human serum and plasma samples

2021 ◽  
Author(s):  
Yongjuan Jia ◽  
Xingli Liu ◽  
Li Xu ◽  
Jinbao Zhao ◽  
Junjun Ni ◽  
...  
Keyword(s):  
Human Serum ◽  
Steroid Hormones ◽  
Plasma Samples

Design and evaluation of standard lipid prediction models based on 1H-NMR spectroscopy of human serum/plasma samples

Metabolomics ◽  
2015 ◽  
Vol 11 (5) ◽  
pp. 1394-1404 ◽  
Author(s):  
Rubén Barrilero ◽  
Eduard Llobet ◽  
Roger Mallol ◽  
Jesús Brezmes ◽  
Lluis Masana ◽  
...  
Keyword(s):  
Nmr Spectroscopy ◽  
Human Serum ◽  
1H Nmr ◽  
1H Nmr Spectroscopy ◽  
Prediction Models ◽  
Plasma Samples

scholarly journals Human Serum Albumin Cys34Oxidative Modifications following Infiltration in the Carotid Atherosclerotic Plaque

2014 ◽  
Vol 2014 ◽  
pp. 1-7 ◽  
Author(s):  
Antonio Junior Lepedda ◽  
Angelo Zinellu ◽  
Gabriele Nieddu ◽  
Pierina De Muro ◽  
Ciriaco Carru ◽  
...  
Keyword(s):  
Human Serum Albumin ◽  
Serum Albumin ◽  
Human Serum ◽  
Carotid Endarterectomy ◽  
Atherosclerotic Plaque ◽  
Plasma Samples ◽  
Carotid Atherosclerotic Plaque ◽  
Sds Page ◽  
Oxidative Modifications ◽  
First Time

Objectives.To evaluate if the prooxidant environment present in atherosclerotic plaque may oxidatively modify filtered albumin.Methods.Fluorescein-5-maleimide labelled plasma samples and plaque extracts from 27 patients who had undergone carotid endarterectomy were analysed through nonreducing SDS-PAGE for albumin-Cys34oxidation. Furthermore, degree and pattern of S-thiolation in both circulating and plaque-filtered albumin were assayed.Results.Albumin filtered in the atherosclerotic plaque showed higher levels of Cys34oxidative modifications than the corresponding circulating form as well as different patterns of S-thiolation.Conclusions.Data indicate that the circulating albumin, once filtered in plaque, undergoes Cys34oxidative modifications and demonstrate for the first time that albumin is a homocysteine and cysteinylglycine vehicle inside the plaque environment.

scholarly journals Monitoring SARS-CoV-2 in sewage: toward sentinels with analytical accuracy

2021 ◽  
Author(s):  
David Calderón-Franco ◽  
Laura Orschler ◽  
Susanne Lackner ◽  
Shelesh Agrawal ◽  
David G. Weissbrodt
Keyword(s):  
Magnetic Beads ◽  
Rna Isolation ◽  
Viral Rna ◽  
Clinical Patient ◽  
Rna Purification ◽  
Viral Particles ◽  
Diethylaminoethyl Cellulose ◽  
Isolation Methods ◽  
Analytical Accuracy ◽  
Epidemiology Studies

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemia has been one of the most difficult challenges humankind has recently faced. Wastewater-based epidemiology has emerged as a tool for surveillance and mitigation of potential viral outbreaks, circumventing biases introduced by clinical patient testing. Due to the situation urgency, protocols followed for isolating viral RNA from sewage were not adapted for such sample matrices. In parallel to their implementation for fast collection of data to sustain surveillance and mitigation decisions, molecular protocols need to be harmonized to deliver accurate, reproducible, and comparable analytical outputs. Here we studied analytical variabilities linked to viral RNA isolation methods from sewage. Three different influent wastewater volumes were used to assess the effect of filtered volumes (50, 100 or 500 mL) for capturing viral particles. Three different concentration strategies were tested by electronegative membranes, polyethersulfone membranes, and anion-exchange diethylaminoethyl cellulose columns. To compare the number of viral particles, different RNA isolation methods (column-based vs. magnetic beads) were compared. The effect of extra RNA purification steps and different RT-qPCR strategies (one step vs. two-step) were also evaluated. Results showed that the combination of 500 mL filtration volume through electronegative membranes and without multiple RNA purification steps (using column-based RNA purification) using two-step RT-qPCR avoided false negatives when basal viral load in sewage are present and yielded more consistent results during the surveillance done during the second-wave in Delft (The Hague area, The Netherlands). By paving the way for standardization of methods for the sampling, concentration and molecular detection of SARS-CoV-2 viruses from sewage, these findings can help water and health surveillance authorities to use and trust results coming from wastewater based epidemiology studies in order to anticipate SARS-CoV-2 outbreaks.

A Radioimmunoassay for Total Captopril in Human Serum or Plasma Samples

1984 ◽  
Vol 6 (1) ◽  
pp. 59-66 ◽  
Author(s):  
J. Tu ◽  
E. Liu ◽  
E. L. Nickoloff
Keyword(s):  
Human Serum ◽  
Plasma Samples

Validation of Isolation Methodology and Endogenous Control Selection for qRT-PCR Assessment of Microrna Expression in Serum and Urine Exosomes

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 5793-5793 ◽  
Author(s):  
Rachel E Crossland ◽  
Jean Norden ◽  
Louis Bibby ◽  
Joanna Davis ◽  
Anne M Dickinson
Keyword(s):  
Electron Microscopy ◽  
Rna Extraction ◽  
Rna Isolation ◽  
Endogenous Control ◽  
Optimal Controls ◽  
Total Rna ◽  
Qrt Pcr ◽  
Rna Purification ◽  
Endogenous Controls ◽  
Exosomal Microrna

Abstract Introduction: MicroRNAs are short RNA molecules that control ~50% of genes by binding to the mRNA 3’ UTR and repressing translation. Recently, they have been detected within exosomes; small vesicles secreted by most cells and abundant in body fluids. Exosomes are highly enriched for specific microRNAs and have been proposed as the starting point for circulating biomarker studies. To increase the accuracy of microRNA assessment by qRT-PCR, endogenous controls are required to correct for variability factors. Exosomal microRNA studies can be problematic, as endogenous controls previously used in cellular samples may not be present. This study compared exosome isolation and RNA extraction methods from urine and serum samples and identified suitable endogenous controls for incorporation into qRT-PCR analysis. Methods and Results: For serum exosomes, specialist isolation reagents from System Biosciences (SBI) (ExoQuick Serum Exosome Precipitation Solution) and Life Technologies (Total Exosome Isolation Reagent) were compared, followed by RNA extraction (Norgen Biotek Total RNA Purification kit) and qRT-PCR assessment of 3 endogenous controls (HY3, RNU48 & U6). Superior exosomal RNA recovery was achieved using Life Technologies reagent, demonstrated by higher RNA concentration (Life Technologies ng/ul 4.4, 7.5 & 6.9 vs. SBI ng/ul 3.8, 5.0 & 2.7) and lower endogenous control Ct values (HY3: Life Technologies 25.56, 28.54 & 26.69 vs. SBI 27.48, 30.48 & 35.36. RNU48: Life Technologies 30.95, undetected & 34.45 vs. SBI 30.95, undetected & undetected. U6: Life Technologies 21.83, 24.72 & 22.59 vs. SBI 21.59, 27.55 & 32.71, respectively). Recovery of exosomes (30-150 nm) was verified by electron microscopy. Serum exosomal RNA recovery was further assessed by isolating exosomes then comparing three commercially available RNA extraction kits (SBI SeraMir Exosome RNA Purification Column kit, Norgen Biotek Total RNA Purification kit & Qiagen RNeasy Micro kit). The Norgen Biotek kit gave the highest RNA yield (SBI ng/ul 13.0, 10.9 & 6.7 vs. Norgen ng/ul 23.2, 22.6 & 33.2 vs. Qiagen ng/ul 0.3, 0.6 & 0.4) and expression of two endogenous controls (HY3 & U6) (HY3: Norgen 26.76, 29.37 & 27.66 vs. SBI 31.45, 29.43 & 33.38 vs. Qiagen 35.00, 35.12 & 33.99. U6: Norgen 21.38, 24.96 & 21.31 vs. SBI 25.95, 24.91 & 30.17 vs. Qiagen 26.48, 27.14 & 27.39). In each case, exosomal isolation was confirmed by electron microscopy. To validate the methodology to isolate urine exosomal RNA, a commercially available kit was compared to ultracentrifugation. The Urine Exosome RNA Isolation kit (Norgen Biotek) gave superior results compared to ultracentrifugation followed by RNA extraction using the Norgen Biotek Total RNA Purification kit. This was demonstrated by higher RNA quantity (Norgen ng/ul 6.6, 6.4 & 11.5 vs. ultracentrifugation ng/ul 3.3, 4.5 & 2.9) and endogenous control (HY3 & U6) expression (HY3: Norgen 25.31, 26.33 & 26.85 vs. ultracentrifugation 31.54, 29.21 & 29.36. U6: Norgen 31.66, 30.83 & 33.47 vs. ultracentrifugation 32.49, 33.46 & 33.30). Exosomes isolated by the Norgen kit were also visualised by electron microscopy for further validation. The stability of 8 endogenous controls (RNU6B, RNU19, RNU38B, RNU43, RNU48, HY3, U6 & miR-320) was assessed by qRT-PCR in a test serum (n=10) and urine (n=15) exosome cohort from healthy controls and hematopoietic stem cell transplantation (HSCT) patients. HY3 and U6 were selected as the optimal controls for serum exosome miRNA expression analysis, with the highest level of stability across the panel (HY3: S.D 1.77 & CoV 6.2%, U6: S.D 2.14 & CoV 8.6%). HY3 and RNU48 were selected as the optimal controls for urine exosome miRNA expression analysis panel (HY3: S.D 1.67 & CoV 6.4%, RNU48: S.D 1.85 & CoV 5.3%). Selected optimal controls were analysed in a clinical HSCT serum (n=55) and urine (n=50) cohort. Expression stability was acceptable for all controls (serum U6: S.D 2.93 & CoV 11.8%. HY3: S.D 2.22 & CoV 7.4%. Urine RNU48: S.D 2.26 & CoV 6.9%, HY3: S.D 2.42 & CoV 8.8%), indicating constitutive expression in clinical samples. Conclusions: Exosomal microRNA studies are in their infancy and the number of commercially available exosome and RNA isolation kits are increasing. This study identifies the optimal methods to isolate serum and urine exosomal RNA as well as suitable endogenous controls for incorporation into qRT-PCR studies. Disclosures No relevant conflicts of interest to declare.

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