Isolation of full-size mRNA from ethanol-fixed cells after cellular immunofluorescence staining and fluorescence-activated cell sorting (FACS)

Cytometry
C EsserA Radbruch

Abstract

Preparation of intact, full-size RNA from tissues or cells requires stringent precautions against ubiquitous and rather stable RNases. Fluorescence-activated cell sorting (FACS) usually aims at the isolation of cells according to cell surface markers on living cells, from which RNA can be obtained by standard protocols. The separation of cells according to intracellular immunofluorescence markers, such as intranuclear, intracytoplasmic, or secreted molecules, requires permeation of the cell membrane for the staining antibodies, which is usually achieved by fixation. However, commonly used fixatives such as ethanol, methanol, or formaldehyde do not inactivate RNases completely, thereby hampering the analysis of complete RNA molecules from fixed cells. We report isolation of intact, full-size RNA suitable for Northern blotting from cells that were fixed by 95% ethanol/5% acetic acid containing RNase inhibitors, stained intracellularly, and sorted by FACS.

References

Apr 12, 1991·Science·D A Melton
Jan 1, 1991·Virchows Archiv. B, Cell Pathology Including Molecular Pathology·C López-GinésA Llombart-Bosch
Jan 1, 1990·Annual Review of Immunology·C Esser, A Radbruch
Jul 16, 1985·Biochemical and Biophysical Research Communications·S E GoelzB Vogelstein
Oct 25, 1988·Nucleic Acids Research·S PääboA C Wilson

❮ Previous
Next ❯

Citations

Sep 16, 1999·Journal of Biochemical and Biophysical Methods·C DiezA Simm
Sep 28, 2002·Journal of Neuroscience Methods·S StollS Thanos
Mar 14, 2013·Biochimica Et Biophysica Acta·Natasha S BartenevaIvan A Vorobjev
Sep 9, 2015·Cytometry. Part a : the Journal of the International Society for Analytical Cytology·Mikael SandstedtJoakim Sandstedt
Jun 2, 2007·Cytometry. Part a : the Journal of the International Society for Analytical Cytology·Ulrik MoerchMartin B Oleksiewicz
May 21, 2017·BMC Biology·Jonathan AllesNikolaus Rajewsky
Dec 22, 2006·Bioorganicheskaia khimiia·A V KravchenkoA B Chetverin
Jul 19, 2018·Journal of Translational Medicine·Jinguo ChenUNKNOWN CHI Consortium
Jan 3, 2012·The Journal of Immunology : Official Journal of the American Association of Immunologists·Leo HansmannMatthias Edinger
Jun 7, 2021·BMC Genomics·Xinlei WangAngela Ruohao Wu

❮ Previous
Next ❯

Related Concepts

Trending Feeds

COVID-19

Coronaviruses encompass a large family of viruses that cause the common cold as well as more serious diseases, such as the ongoing outbreak of coronavirus disease 2019 (COVID-19; formally known as 2019-nCoV). Coronaviruses can spread from animals to humans; symptoms include fever, cough, shortness of breath, and breathing difficulties; in more severe cases, infection can lead to death. This feed covers recent research on COVID-19.

Blastomycosis

Blastomycosis fungal infections spread through inhaling Blastomyces dermatitidis spores. Discover the latest research on blastomycosis fungal infections here.

Nuclear Pore Complex in ALS/FTD

Alterations in nucleocytoplasmic transport, controlled by the nuclear pore complex, may be involved in the pathomechanism underlying multiple neurodegenerative diseases including Amyotrophic Lateral Sclerosis and Frontotemporal Dementia. Here is the latest research on the nuclear pore complex in ALS and FTD.

Applications of Molecular Barcoding

The concept of molecular barcoding is that each original DNA or RNA molecule is attached to a unique sequence barcode. Sequence reads having different barcodes represent different original molecules, while sequence reads having the same barcode are results of PCR duplication from one original molecule. Discover the latest research on molecular barcoding here.

Chronic Fatigue Syndrome

Chronic fatigue syndrome is a disease characterized by unexplained disabling fatigue; the pathology of which is incompletely understood. Discover the latest research on chronic fatigue syndrome here.

Evolution of Pluripotency

Pluripotency refers to the ability of a cell to develop into three primary germ cell layers of the embryo. This feed focuses on the mechanisms that underlie the evolution of pluripotency. Here is the latest research.

Position Effect Variegation

Position Effect Variagation occurs when a gene is inactivated due to its positioning near heterochromatic regions within a chromosome. Discover the latest research on Position Effect Variagation here.

STING Receptor Agonists

Stimulator of IFN genes (STING) are a group of transmembrane proteins that are involved in the induction of type I interferon that is important in the innate immune response. The stimulation of STING has been an active area of research in the treatment of cancer and infectious diseases. Here is the latest research on STING receptor agonists.

Microbicide

Microbicides are products that can be applied to vaginal or rectal mucosal surfaces with the goal of preventing, or at least significantly reducing, the transmission of sexually transmitted infections. Here is the latest research on microbicides.

Related Papers

Journal of Biochemical and Biophysical Methods
C DiezA Simm
Cytometry. Part a : the Journal of the International Society for Analytical Cytology
Hiroya YamadaToru Takano
Journal of Bone and Mineral Research : the Official Journal of the American Society for Bone and Mineral Research
Clifford J Rosen
© 2021 Meta ULC. All rights reserved