scholarly journals Characterization of the Impact of Density Gradient Centrifugation on the Profile of the Pig Sperm Transcriptome by RNA-Seq

2021 ◽  
Vol 8 ◽  
Author(s):  
Yu Lian ◽  
Marta Gòdia ◽  
Anna Castello ◽  
Joan Enric Rodriguez-Gil ◽  
Sam Balasch ◽  
...  
Keyword(s):  
Density Gradient ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Principal Component ◽  
Rna Seq ◽  
Protein Coding ◽  
The Impact ◽  
Germinal Cells ◽  
Rna Levels

RNA-Seq data from human semen suggests that the study of the sperm transcriptome requires the previous elimination from the ejaculates of somatic cells carrying a larger load of RNA. Semen purification is also carried to study the sperm transcriptome in other species including swine and it is often done by density gradient centrifugation to obtain viable spermatozoa from fresh ejaculates or artificial insemination doses, thereby limiting the throughput and remoteness of the samples that can be processed in one study. The aim of this work was to evaluate the impact of purification with density gradient centrifugation by BoviPureTM on porcine sperm. Four boar ejaculates were purified with BoviPureTM and their transcriptome sequenced by RNA-Seq was compared with the RNA-Seq profiles of their paired non-purified sample. Seven thousand five hundred and nineteen protein coding genes were identified. Correlation, cluster, and principal component analysis indicated high—although not complete—similarity between the purified and the paired non-purified ejaculates. 372 genes displayed differentially abundant RNA levels between treatments. Most of these genes had lower abundances after purification and were mostly related to translation, transcription and metabolic processes. We detected a significant change in the proportion of genes of epididymal origin within the differentially abundant genes (1.3%) when compared with the catalog of unaltered genes (0.2%). In contrast, the proportion of testis-specific genes was higher in the group of unaltered genes (4%) when compared to the list of differentially abundant genes (0%). No proportion differences were identified for prostate, white blood, lymph node, tonsil, duodenum, skeletal muscle, liver, and mammary gland. Altogether, these results suggest that the purification impacts on the RNA levels of a small number of genes which are most likely caused by the removal of epididymal epithelial cells but also premature germinal cells, immature or abnormal spermatozoa or seminal exosomes with a distinct load of RNAs.

scholarly journals Isolation and characterization of human cervical-mucus glycoproteins

1983 ◽  
Vol 211 (1) ◽  
pp. 13-22 ◽  
Author(s):  
I Carlstedt ◽  
H Lindgren ◽  
J K Sheehan ◽  
U Ulmsten ◽  
L Wingerup
Keyword(s):  
Density Gradient ◽  
Proteinase Inhibitors ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Disc Electrophoresis ◽  
Guanidinium Chloride ◽  
Molecular Weights ◽  
Isolation And Characterization ◽  
Mucus Glycoproteins

Mucus glycoproteins (mucins) were extracted from human cervical pregnancy mucus by 6 M-guanidinium chloride in the presence of proteinase inhibitors. Purification was subsequently achieved by isopycnic density-gradient centrifugation in CsCl/ guanidinium chloride gradients. The purified macromolecules represented approx. 85% of the total and were devoid of nucleic acids and proteins, as judged by analytical density-gradient centrifugation, disc electrophoresis and u.v. spectroscopy. Sedimentation-velocity centrifugation revealed a single unimodal peak with S20,W 50.1S in 0.2M-NaCl and 37.0S in 6 M-guanidinium chloride. Molecular weights obtained by light-scattering were 9.7 × 10(6) and 5.9 × 10(6) in 0.2M-NaCl and 6 M-guanidinium chloride respectively. The chemical analyses were typical of those of epithelial mucins. The macromolecules contained approx. 20% (w/w) of protein, and 65% (w/w) was accounted for as carbohydrate. Serine and threonine constituted 32 mol/100 mol and proline 10 mol/100 mol of the amino acids. The major sugars found were N-acetylglucosamine (12.8%), N-acetylgalactosamine (9.7%), galactose (18.7%), sialic acid (15.0%) and fucose (7.5%).

scholarly journals Delays during PBMC isolation have a moderate effect on yield, but severly compromise cell viability

2022 ◽  
Author(s):  
Tanja Golke ◽  
Patrick Mucher ◽  
Patricia Schmidt ◽  
Astrid Radakovics ◽  
Manuela Repl ◽  
...  
Keyword(s):  
Cell Viability ◽  
Density Gradient ◽  
Standard Method ◽  
Mononuclear Cells ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Initial Density ◽  
Minor Effect ◽  
Peripheral Blood Mononuclear ◽  
The Impact

Background: Peripheral blood mononuclear cells (PMBCs) are a versatile material for clinical routine as well as for research projects. However, their isolation via density gradient centrifugation is still time-consuming. When samples are taken beyond usual laboratory handling times, it may sometimes be necessary to pause the isolation process. Our aim was to evaluate the impact of delays up to 48 hours after the density gradient centrifugation on PBMC yield, purity and viability. Methods: PBMCs were isolated from samples of 20 donors, either with BD Vacutainer CPT tubes (CPT) or with the standard Ficoll method. Isolation was paused after initial density gradient centrifugation for 0, 24, or 48 hours. PBMC yield, purity and viability were compared. Results: The yield did not change significantly over time when CPT were used (55%/52%/47%), but did after isolation with the standard method (62%/40%[p<0.0001]/53%[p<0.01]). Purity was only affected if CPT were used (95%/93%[p=n.s./92%[p<0.05] vs. 97% for all time points with standard method). Whereas viable PBMCs decreased steadily for CPT isolates (62%/51%[p<0.001]/36%[p<0.0001]), after standard Ficoll gradient isolation, cell apoptosis was more pronounced already after 24h delay, and viability did not further decrease after 48h (64%/44%[p<0.0001]/40%[p<0.0001]). Conclusions: In conclusion, our data suggests that post-centrifugation delays of up to 48h might have only a minor effect on cell yield and purity. However, at the same time, a relevant decrease in cell viability was observed, which could be partially compensated by the use of CPT if the isolation was resumed latest the day after blood withdrawal.

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