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Quantitative single-cell RNA-sequencing for alcohol- and formalin-fixed tissue sections with DRaqL; an efficient RNA recovery and cDNA amplification method for laser capture microdissection

ABSTRACT: Spatially resolved, single-cell transcriptome analysis of high quality remains challenging, despite the rise of high-resolution spatial transcriptomics. Laser-capture microdissection (LCM) is widely used to isolate cells of interest from tissue sections. Here, we developed DRaqL (Direct RNA recovery and quenching for LCM), a technique that allows efficient lysis of single, LCM-isolated cells from alcohol- and formalin-fixed tissue and stained frozen sections, and that is amenable to enzymatic reactions for cDNA amplification within the same sampling tubes. Quantitative evaluations showed that single-cell RNA sequencing combined with DRaqL allowed transcriptomic profiling from frozen sections at an efficiency comparable with that from freshly dissociated cells, with small biases and errors in lowly expressed genes, in addition to allowing exon-exon junction profiling. By applying this method to mouse ovarian frozen sections, we revealed a transcriptomic continuum of growing oocytes quantitatively associated with the size of oocytes and follicles, identified genes highly correlated with oocyte diameters, and detected oocyte-specific splice isoforms. We further identified genes that were differentially expressed in granulosa cells depending on their distance from oocytes, suggesting distinct epigenetic regulatory mechanisms and cellular proliferation. Thus, DRaqL provides an efficient cell lysis for single-cell cDNA amplification from frozen sections that is amenable to high-quality RNA sequencing analysis.

ORGANISM(S): Mus musculus

PROVIDER: GSE192551 | GEO | 2023/09/01

REPOSITORIES: GEO

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Project description:Study question: Does storage time impact on transcriptome of slowly frozen cryopreserved human metaphase II (MII) oocytes? Summary answer: For the first time, we demonstrate that the length of cryostorage has no effect on the gene expression profile of human metaphase II oocytes. What is known already: Oocyte cryopreservation is a largely-used technique in IVF for storage of surplus oocytes, as well as for fertility preservation (i.e., women undergoing gonadotoxic therapies) and oocyte donation programs. Although it is known that cryopreservation negatively impacts on oocyte physiology and it is associated with decrease of transcripts, no experimental data about the effect of storage time on the oocyte molecular profile are available to date. Study design, size, duration: This study included 27 women undergoing IVF treatment, < 38 years aged, without any ovarian pathology. Surplus MII oocytes were donated after written informed consent. A total of 31 non-cryopreserved oocytes and 68 surviving slow-frozen/rapid-thawed oocytes (32 oocytes cryostored for 3 years and 36 cryostored for 6 years) were analyzed. Participants/materials, setting, methods: Pools of ?10 oocytes for each group were prepared. Total RNA was extracted from each pool, amplified, labeled and hybridized on 4x44K v2 microarrays (Agilent). Analyses were performed by R v3.1.3 software using the limma package v3.22.7. Main results and the role of chance: Comparison of gene expression profiles between surviving thawed oocytes after 3 and 6 years of storage in liquid nitrogen found no differently expressed genes. The expression profiles of cryopreserved MII oocytes significantly differed from those of non-cryopreserved oocytes in 107 probe sets corresponding to 73 down-regulated and 29 up-regulated unique transcripts. Gene Ontology analysis by DAVID bioinformatics resource disclosed that cryopreservation deregulates genes involved in oocyte function and early embryo development, such as chromosome organization, RNA splicing and processing, cell cycle process, response to DNA damage stimulus, cellular response to stress and DNA repair, calcium ion binding, malate dehydrogenase activity, mitochondrial membrane respiratory chain. Among the probes significantly up-regulated in cryopreserved oocytes, 2 corresponded to ovary-specific expressed large intergenic noncoding (linc)RNAs. This study included 27 women undergoing IVF treatment, < 38 years aged, without any ovarian pathology. Surplus MII oocytes were donated after written informed consent. A total of 31 non-cryopreserved oocytes and 68 surviving slow-frozen/rapid-thawed oocytes (32 oocytes cryostored for 3 years and 36 cryostored for 6 years) were analyzed.

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Quantitative single-cell RNA-sequencing for alcohol- and formalin-fixed tissue sections with DRaqL; an efficient RNA recovery and cDNA amplification method for laser capture microdissection

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