Buffer A (Hypotonic Lysis Buffer)

2019 ◽  
Vol 2019 (7) ◽  
pp. pdb.rec102004
Keyword(s):  
Hypotonic Lysis ◽  
Lysis Buffer

Hypotonic lysis buffer

2007 ◽  
Vol 2007 (7) ◽  
pp. pdb.rec10775-pdb.rec10775
Keyword(s):  
Hypotonic Lysis ◽  
Lysis Buffer

scholarly journals Self-Collection of Saliva Specimens as a Suitable Alternative to Nasopharyngeal Swabs for the Diagnosis of SARS-CoV-2 by RT-qPCR

2021 ◽  
Vol 10 (2) ◽  
pp. 299
Author(s):  
Camino Trobajo-Sanmartín ◽  
Marta Adelantado ◽  
Ana Navascués ◽  
María J. Guembe ◽  
Isabel Rodrigo-Rincón ◽  
...  
Keyword(s):  
Chronic Conditions ◽  
Saliva Sample ◽  
Virus Detection ◽  
Kappa Coefficient ◽  
Nasopharyngeal Swab ◽  
Personal Protection ◽  
Personal Protection Equipment ◽  
Suitable Alternative ◽  
Lysis Buffer ◽  
Sensitivity Specificity

A nasopharyngeal swab is a sample used for the diagnosis of SARS-CoV-2 infection. Saliva is a sample easier to obtain and the risk of contagion for the professional is lower. This study aimed to evaluate the utility of saliva for the diagnosis of SARS-CoV-2 infection. This prospective study involved 674 patients with suspected SARS-CoV-2 infection. Paired nasopharyngeal and saliva samples were processed by RT-qPCR. Sensitivity, specificity, and kappa coefficient were used to evaluate the results from both samples. We considered the influence of age, symptoms, chronic conditions, and sample processing with lysis buffer. Of the 674 patients, 636 (94.4%) had valid results from both samples. The virus detection in saliva compared to a nasopharyngeal sample (gold standard) was 51.9% (95% CI: 46.3%–57.4%) and increased to 91.6% (95% CI: 86.7%–96.5%) when the cycle threshold (Ct) was ≤ 30. The specificity of the saliva sample was 99.1% (95% CI: 97.0%–99.8%). The concordance between samples was 75% (κ = 0.50; 95% CI: 0.45–0.56). The Ct values were significantly higher in saliva. In conclusion, saliva sample utility is limited for clinical diagnosis, but could be a useful alternative for the detection of SARS-CoV-2 in massive screening studies, when the availability of trained professionals for sampling or personal protection equipment is limited.

scholarly journals Importance of experimental information (metadata) for archived sequence data: case of specific gene bias due to lag time between sample harvest and RNA protection in RNA sequencing

PeerJ ◽  
2021 ◽  
Vol 9 ◽  
pp. e11875
Author(s):  
Tomoko Matsuda
Keyword(s):  
Gene Expression ◽  
Rna Sequencing ◽  
Time Course ◽  
Sequence Data ◽  
Specific Gene ◽  
Time Interval ◽  
Short Time Interval ◽  
Rna Seq ◽  
Lysis Buffer ◽  
Rna Protection

Large volumes of high-throughput sequencing data have been submitted to the Sequencing Read Archive (SRA). The lack of experimental metadata associated with the data makes reuse and understanding data quality very difficult. In the case of RNA sequencing (RNA-Seq), which reveals the presence and quantity of RNA in a biological sample at any moment, it is necessary to consider that gene expression responds over a short time interval (several seconds to a few minutes) in many organisms. Therefore, to isolate RNA that accurately reflects the transcriptome at the point of harvest, raw biological samples should be processed by freezing in liquid nitrogen, immersing in RNA stabilization reagent or lysing and homogenizing in RNA lysis buffer containing guanidine thiocyanate as soon as possible. As the number of samples handled simultaneously increases, the time until the RNA is protected can increase. Here, to evaluate the effect of different lag times in RNA protection on RNA-Seq data, we harvested CHO-S cells after 3, 5, 6, and 7 days of cultivation, added RNA lysis buffer in a time course of 15, 30, 45, and 60 min after harvest, and conducted RNA-Seq. These RNA samples showed high RNA integrity number (RIN) values indicating non-degraded RNA, and sequence data from libraries prepared with these RNA samples was of high quality according to FastQC. We observed that, at the same cultivation day, global trends of gene expression were similar across the time course of addition of RNA lysis buffer; however, the expression of some genes was significantly different between the time-course samples of the same cultivation day; most of these differentially expressed genes were related to apoptosis. We conclude that the time lag between sample harvest and RNA protection influences gene expression of specific genes. It is, therefore, necessary to know not only RIN values of RNA and the quality of the sequence data but also how the experiment was performed when acquiring RNA-Seq data from the database.

Studies of the endoplasmic reticulum and plasma membrane-bound ribosomes in erythropoietic cells

1978 ◽  
Vol 31 (1) ◽  
pp. 165-178
Author(s):  
J.A. Grasso ◽  
A.L. Sullivan ◽  
S.C. Chan
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Exact Role ◽  
Whole Cells ◽  
Cytoplasmic Face ◽  
Close Proximity ◽  
Haem Biosynthesis ◽  
Hypotonic Buffer ◽  
Membrane Bound ◽  
Hypotonic Lysis

Erythropoietic cells of 5 species, including man, contain endoplasmic reticulum present as individual cisternae or tubules scattered throughout the cytoplasm of all stages except mature RBCs. The endoplasmic reticulum is mainly agranular but occurs frequently as a variant of granular ER which is characterized by an asymmetrical and irregular distribution of ribosomes along one cytoplasmic face. In most cells, the endoplasmic reticulum occurs in close proximity to mitochondria or the plasma membrane, suggesting that the organelle may be involved in functions related to these structures, e.g. haem biosynthesis. Endoplasmic reticulum is more abundant in early than in late erythroid cells. Its exact role in RBC development is unclear. Since endoplasmic reticulum could account for ‘plasma membrane-bound ribosomes’ reported in lysed reticulocytes, studies were performed which ruled out this possibility and which suggested that such ribosomes were an artifact of the lysing conditions. Hypotonic lysis in less than 20 vol. of magnesium-containing buffers yielded ghosts variably contaminated by ribosomes and other structures. Lysis of reticulocytes in 20–30 vol. of magnesium-free buffer or homogenization of whole cells or crude membrane fractions in hypotonic buffer removed virtually all contaminating ribosomes from the purified membrane fraction.

Picoliter liquid handling at gas/liquid interface by surface and geometry control in a micro-nanofluidic device

2021 ◽  
Author(s):  
Kyojiro Morikawa ◽  
Shin-ichi Murata ◽  
Y Kazoe ◽  
Kazuma Mawatari ◽  
Takehiko Kitamori
Keyword(s):  
Surface Tension ◽  
Single Cell ◽  
Single Cell Analysis ◽  
Cell Lysis ◽  
Fused Silica ◽  
Liquid Interface ◽  
Nanofluidic Devices ◽  
Fluidic Control ◽  
Lysis Buffer ◽  
Nanofluidic Device

Abstract In micro- and nanofluidic devices, highly precise fluidic control is essential. Conventional mechanical valves in microchannels and nanochannels have size limitations, whereas hydrophobic (Laplace) valves are generally difficult to use for low-surface-tension liquids. In the present study, we developed a method for handling picoliter volumes of low-surface-tension liquids in a micro-nanofluidic device. The proposed Laplace valve is based on the pinning effect. A fused silica micro-nanofluidic device that includes a picoliter chamber whose geometry was designed to induce capillary pinning was designed and fabricated. The measured Laplace pressure of a lysis buffer (surfactant) was consistent with the calculated pressure, indicating successful fabrication and hydrophobic surface modification. The working principle of the Laplace valve was verified. The Laplace valve maintained the lysis buffer at the gas/liquid interface for 60 min, which is sufficiently long for cell lysis operations. Finally, replacement of liquids in the picoliter chamber using the valve was demonstrated. The proposed method will contribute to basic technologies for fluidic control in micro- and nanofluidic devices, and the proposed Laplace valve can be used for low-surface-tension liquids. In addition, the developed valve and picoliter chamber can be utilized for the interface in single-cell lysis, which will facilitate the development of single-cell analysis devices.

scholarly journals Endoplasmic Reticulum Detergent-Resistant Membranes Accommodate Hepatitis C Virus Proteins for Viral Assembly

Cells ◽  
2019 ◽  
Vol 8 (5) ◽  
pp. 487 ◽  
Author(s):  
Audrey Boyer ◽  
Julie Dreneau ◽  
Amélie Dumans ◽  
Julien Burlaud-Gaillard ◽  
Anne Bull-Maurer ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Protein Interactions ◽  
Gradient Centrifugation ◽  
Cellular Membrane ◽  
Structural Protein ◽  
Membrane Structures ◽  
Detergent Resistant Membranes ◽  
Lysis Buffer

During Hepatitis C virus (HCV) morphogenesis, the non-structural protein 2 (NS2) brings the envelope proteins 1 and 2 (E1, E2), NS3, and NS5A together to form a complex at the endoplasmic reticulum (ER) membrane, initiating HCV assembly. The nature of the interactions in this complex is unclear, but replication complex and structural proteins have been shown to be associated with cellular membrane structures called detergent-resistant membranes (DRMs). We investigated the role of DRMs in NS2 complex formation, using a lysis buffer combining Triton and n-octyl glucoside, which solubilized both cell membranes and DRMs. When this lysis buffer was used on HCV-infected cells and the resulting lysates were subjected to flotation gradient centrifugation, all viral proteins and DRM-resident proteins were found in soluble protein fractions. Immunoprecipitation assays demonstrated direct protein–protein interactions between NS2 and E2 and E1 proteins, and an association of NS2 with NS3 through DRMs. The well-folded E1E2 complex and NS5A were not associated, instead interacting separately with the NS2-E1-E2-NS3 complex through less stable DRMs. Core was also associated with NS2 and the E1E2 complex through these unstable DRMs. We suggest that DRMs carrying this NS2-E1-E2-NS3-4A-NS5A-core complex may play a central role in HCV assembly initiation, potentially as an assembly platform.

Higher Cardol Homologues (5-Alkenylresorcinols) from Rye Affect the Red Cell Membrane-Water Transport

1985 ◽  
Vol 40 (1-2) ◽  
pp. 80-84 ◽  
Author(s):  
Arkadiusz Kozubek
Keyword(s):  
Cell Membrane ◽  
Blood Cell ◽  
Water Permeability ◽  
Threshold Concentration ◽  
Water Soluble ◽  
Red Cell ◽  
Cell Water ◽  
Red Cell Membrane ◽  
Soluble Surfactants ◽  
Hypotonic Lysis

The influence of 5-heptadecenylresorcinol and total rye 5-alkenylresorcinols isolated from rye grains on the red blood cell water permeability was studied using osmotic shrinkage experiments performed in 300 mᴍ sucrose. The studied compounds induced significant increase of erythro­cyte water permeability. The threshold concentration needed for the increase of water per­meability was in an order of 10-6mol/l. The temperature dependence of the observed process showed the discontinuity which was related to the 5-alkenylresorcinol transition temperatures. It was shown also that alkenylresorcinols did not exert the biphasic action on hypotonic lysis of erythrocytes usually observed for water soluble surfactants. The specific lysine activity is postulated for the studied compounds.

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