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

Kimble, Katelyn M.; Dickinson, Sarah; Biase, Fernando H.

doi:10.1186/s13104-018-3264-2

Cited by 3 publications

(3 citation statements)

References 17 publications

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“…RNA extracted from IRF-0399 cultures was used for developing and optimizing singleplex and duplex RT-qPCR assays. The RNA extraction method was based on earlier published methods with some modifications [ 40 ]. Briefly, a volume of 0.75 mL of TRIzol LS reagent (Thermo Fisher Scientific) was added to 0.25 mL of Vero cells supernatants, and samples were transferred to Invitrogen Phasemaker Tubes (Thermo Fisher Scientific).…”

Section: Methodsmentioning

confidence: 99%

Duplex One-Step RT-qPCR Assays for Simultaneous Detection of Genomic and Subgenomic RNAs of SARS-CoV-2 Variants

Bhosle

Tran

Yú

et al. 2022

Viruses

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A hallmark of severe acute respiratory syndrome virus (SARS-CoV-2) replication is the discontinuous transcription of open reading frames (ORFs) encoding structural virus proteins. Real-time reverse transcription PCR (RT-qPCR) assays in previous publications used either single or multiplex assays for SARS-CoV-2 genomic RNA detection and a singleplex approach for subgenomic RNA detection. Although multiplex approaches often target multiple genomic RNA segments, an assay that concurrently detects genomic and subgenomic targets has been lacking. To bridge this gap, we developed two duplex one-step RT-qPCR assays that detect SARS-CoV-2 genomic ORF1a and either subgenomic spike or subgenomic ORF3a RNAs. All primers and probes for our assays were designed to bind to variants of SARS-CoV-2. In this study, our assays successfully detected SARS-CoV-2 Washington strain and delta variant isolates at various time points during the course of live virus infection in vitro. The ability to quantify subgenomic SARS-CoV-2 RNA is important, as it may indicate the presence of active replication, particularly in samples collected longitudinally. Furthermore, specific detection of genomic and subgenomic RNAs simultaneously in a single reaction increases assay efficiency, potentially leading to expedited lucidity about viral replication and pathogenesis of any variant of SARS-CoV-2.

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Section: Methodsmentioning

confidence: 99%

Duplex One-Step RT-qPCR Assays for Simultaneous Detection of Genomic and Subgenomic RNAs of SARS-CoV-2 Variants

Bhosle

Tran

Yú

et al. 2022

Viruses

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“…For each of the 16 COCs, we prepared libraries for the oocyte, innerCC and outerCC cells. We extracted RNA from cumulus cells using the guanidine thiocyanate-phenol chloroform procedure [ 38 , 39 ] (TRIzol reagent, Thermo Fisher), with the addition of 0.5 μl of GlycoBlue Coprecipitant (Thermo Fisher) as the RNA carrier [ 40 ]. We eluted the RNA from CCs in 4 μl of a solution containing dNTPs and oligo-dT 30 VN and proceeded with polyA+ whole transcriptome amplification using the SMART-seq2 protocol [ 41 , 42 ] for cDNA amplification.…”

Section: Methodsmentioning

confidence: 99%

Functional signaling and gene regulatory networks between the oocyte and the surrounding cumulus cells

Biase

Kimble

2018

BMC Genomics

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BackgroundThe maturation and successful acquisition of developmental competence by an oocyte, the female gamete, during folliculogenesis is highly dependent on molecular interactions with somatic cells. Most of the cellular interactions identified, thus far, are modulated by growth factors, ions or metabolites. We hypothesized that this interaction is also modulated at the transcriptional level, which leads to the formation of gene regulatory networks between the oocyte and cumulus cells. We tested this hypothesis by analyzing transcriptome data from single oocytes and the surrounding cumulus cells collected from antral follicles employing an analytical framework to determine interdependencies at the transcript level.ResultsWe overlapped our transcriptome data with putative protein-protein interactions and identified hundreds of ligand-receptor pairs that can transduce paracrine signaling between an oocyte and cumulus cells. We determined that 499 ligand-encoding genes expressed in oocytes and cumulus cells are functionally associated with transcription regulation (FDR < 0.05). Ligand-encoding genes with specific expression in oocytes or cumulus cells were enriched for biological functions that are likely associated with the coordinated formation of transzonal projections from cumulus cells that reach the oocyte’s membrane. Thousands of gene pairs exhibit significant linear co-expression (absolute correlation > 0.85, FDR < 1.8 × 10− 5) patterns between oocytes and cumulus cells. Hundreds of co-expressing genes showed clustering patterns associated with biological functions (FDR < 0.5) necessary for a coordinated function between the oocyte and cumulus cells during folliculogenesis (i.e. regulation of transcription, translation, apoptosis, cell differentiation and transport).ConclusionOur analyses revealed a complex and functional gene regulatory circuit between the oocyte and surrounding cumulus cells. The regulatory profile of each cumulus-oocyte complex is likely associated with the oocytes’ developmental potential to derive an embryo.Electronic supplementary materialThe online version of this article (10.1186/s12864-018-4738-2) contains supplementary material, which is available to authorized users.

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“…Due to small sample volumes, many oocyte-focused studies rely on pooled oocyte samples to have adequate substrate to perform analyses. As technology has advanced, single cell oocyte analysis is possible [38]. Because ATP concentration and mitochondrial numbers within oocytes are important for downstream developmental competence, it is integral that protocols are developed to further investigate these parameters within single oocytes.…”

Section: Discussionmentioning

confidence: 99%

Concurrent Measurement of Mitochondrial DNA Copy Number and ATP Concentration in Single Bovine Oocytes

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Bhandari

Moorey

2021

MPs

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To sustain energy-demanding developmental processes, oocytes must accumulate adequate stores of metabolic substrates and mitochondrial numbers prior to the initiation of maturation. In the past, researchers have utilized pooled samples to study oocyte metabolism, and studies that related multiple metabolic outcomes in single oocytes, such as ATP concentration and mitochondrial DNA copy number, were not possible. Such scenarios decreased sensitivity to intraoocyte metabolic relationships and made it difficult to obtain adequate sample numbers during studies with limited oocyte availability. Therefore, we developed and validated procedures to measure both mitochondrial DNA (mtDNA) copy number and ATP quantity in single oocytes. Validation of our procedures revealed that we could successfully divide oocyte lysates into quarters and measure consistent results from each of the aliquots for both ATP and mtDNA copy number. Coefficient of variation between the values retrieved for mtDNA copy number and ATP quantity quadruplicates were 4.72 ± 0.98 and 1.61 ± 1.19, respectively. We then utilized our methodology to concurrently measure mtDNA copy number and ATP quantity in germinal vesicle (GV) and metaphase two (MII) stage oocytes. Our methods revealed a significant increase in ATP levels (GV = 628.02 ± 199.53 pg, MII = 1326.24 ± 199.86 pg, p < 0.001) and mtDNA copy number (GV = 490,799.4 ± 544,745.9 copies, MII = 1,087,126.9 ± 902,202.8 copies, p = 0.035) in MII compared to GV stage oocytes. This finding is consistent with published literature and provides further validation of the accuracy of our methods. The ability to produce consistent readings and expected results from aliquots of the lysate from a single oocyte reveals the sensitivity and feasibility of using this method.

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

Cited by 3 publications

References 17 publications

Duplex One-Step RT-qPCR Assays for Simultaneous Detection of Genomic and Subgenomic RNAs of SARS-CoV-2 Variants

Duplex One-Step RT-qPCR Assays for Simultaneous Detection of Genomic and Subgenomic RNAs of SARS-CoV-2 Variants

Functional signaling and gene regulatory networks between the oocyte and the surrounding cumulus cells

Concurrent Measurement of Mitochondrial DNA Copy Number and ATP Concentration in Single Bovine Oocytes

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