Extraction of total RNA from single-oocytes and single-cell mRNA sequencing of swine oocytes

BMC Research Notes
Katelyn M KimbleFernando H Biase

Abstract

Analyses of single oocytes are essential for a fine dissection of molecular features governing developmental competence. We adapted the phenol-chloroform procedure for the purification of total RNA from single oocytes. Key modifications include the use of Phasemaker™ tubes, a second chloroform wash of the aqueous phase, and the precipitation of the RNA with glyclogen in a 200 μl micro-centrifuge tube. Assessment of the RNA profile from single oocytes showed distinct peaks for 18S and 28S ribosomal subunits. This approach permitted the extraction of small RNAs from single oocytes, which was evident by the presence of 5S and 5.8S rRNAs and tRNAs around 122-123 nucleotides long. The amplification of polyadenylated RNA resulted in detectable DNA products ranging from ~ 500 to ~ 5000 nucleotides. We used the amplified DNA as template for single-cell mRNA-sequencing of five swine oocytes and quantified the expression levels of 9587 genes with complete coverage of transcripts over 10,000 nucleotides in length. The coverage was similar in all oocytes sequenced, demonstrating consistent high RNA quality across samples. We isolated total RNA from single oocytes and demonstrated that the quality was appropriate for single-cell mRNA-sequen...Continue Reading

References

Feb 17, 2009·Biochemistry. Biokhimii︠a︡·A V SmirnovI A Tarassov
Apr 8, 2009·Nature Methods·Fuchou TangM Azim Surani
Oct 13, 2009·Fertility and Sterility·Fernando Henrique BiaseFlávio Vieira Meirelles
Dec 1, 2011·Genome Research·Jason GertzRichard M Myers
Mar 6, 2012·Nature Methods·Ben Langmead, Steven L Salzberg
Jul 4, 2012·Journal of Applied Microbiology·C SchraderR Johne
Oct 11, 2012·Zygote : the Biology of Gametes and Early Embryos·Fernando Henrique BiaseFlávio Vieira Meirelles
Nov 20, 2013·BMC Research Notes·Susanna Cirera
Jan 5, 2014·Nature Protocols·Simone PicelliRickard Sandberg
Jan 7, 2015·Molecular Reproduction and Development·Juan M ReyesPablo J Ross

❮ Previous
Next ❯

Citations


❮ Previous
Next ❯

Methods Mentioned

BETA
dissection
single-cell sequencing

Software Mentioned

Bowtie2
R
Ensembl

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.