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BackgroundOogenesis in the domestic silkworm (Bombyx mori) is a complex process involving previtellogenesis, vitellogenesis and choriogenesis. During this process, follicles show drastic morphological and physiological changes. However, the genome-wide regulatory profiles of gene expression during oogenesis remain to be determined.ResultsIn this study, we obtained time-series transcriptome data and used these data to reveal the dynamic landscape of gene regulation during oogenesis. A total of 1932 genes were identified to be differentially expressed among different stages, most of which occurred during the transition from late vitellogenesis to early choriogenesis. Using weighted gene co-expression network analysis, we identified six stage-specific gene modules that correspond to multiple regulatory pathways. Strikingly, the biosynthesis pathway of the molting hormone 20-hydroxyecdysone (20E) was enriched in one of the modules. Further analysis showed that the ecdysteroid 20-hydroxylase gene (CYP314A1) of steroidgenesis genes was mainly expressed in previtellogenesis and early vitellogenesis. However, the 20E–inactivated genes, particularly the ecdysteroid 26-hydroxylase encoding gene (Cyp18a1), were highly expressed in late vitellogenesis. These distinct expression patterns between 20E synthesis and catabolism-related genes might ensure the rapid decline of the hormone titer at the transition point from vitellogenesis to choriogenesis. In addition, we compared landscapes of gene regulation between silkworm (Lepidoptera) and fruit fly (Diptera) oogeneses. Our results show that there is some consensus in the modules of gene co-expression during oogenesis in these insects.ConclusionsThe data presented in this study provide new insights into the regulatory mechanisms underlying oogenesis in insects with polytrophic meroistic ovaries. The results also provide clues for further investigating the roles of epigenetic reconfiguration and circadian rhythm in insect oogenesis.Electronic supplementary materialThe online version of this article (10.1186/s12864-017-4123-6) contains supplementary material, which is available to authorized users.

In this work, we present a simple and equipment-free system for discretizing samples into tens of thousands of discrete volumes in tens of seconds. Unlike conventional sample discretization systems that...

known for decades, its widespread use in biology and medicine remains limited due to the lack of simple and high-throughput methods for the production of high-quality spheroids. To date, a lot of methods have been suggested for producing cell spheroids, the earliest of which are mainly based on spinning flasks and rotating reactors. [2] Although these methods can massively produce spheroids, they have critical limitations in reproducibility and spheroid uniformity, and are also liable to cause shear damage to spheroids. Moreover, it is difficult with these methods to monitor the change of individual spheroids during culture. As a consequence, the hanging drop (HD) method was subsequently proposed for cell spheroid generation, where medium droplets containing cell suspensions are hanging from inverted substrate surfaces and gravitational force induces cell aggregation at the liquid-air interface. [3][4][5] Such a feature of cell culture on a curved liquid-air interface provides several distinct advantages, such as simple operation, high oxygen availability to cell aggregations, and improved performance for spheroid uniformity. Although these advantages make the HD method one of the most widely used approaches for spheroid formation, it suffers from inherent instability of the hanging droplets and difficulty in exchanging medium, limiting its use to short-term spheroid cultures. [6] Recently, considerable efforts have been made to develop improved spheroid production technologies, including acoustic-wave-actuated aggregation, [7,8] magnetic levitation, [9] emulsion-based encapsulation, [10] liquid marble-based encapsulation, [11] and non-adherent microwells. [12] Although these methods provide certain advantages such as high-throughput of spheroid generation or simplicity of liquid handling, most of them are technically complicated, costly, and require sophisticated equipment or specialized training. Worse yet, the availability of oxygen to spheroidal cellular aggregations cultured in these platforms is often poor, which is detrimental to cell viability and spheroid functionality. [13] In this work, we propose a novel cell culture strategy to address many limitations of current platforms. In contrast to conventional HD methods, this strategy makes use of a superhydrophobic perforated microwell plate (SHPMP) to culture cell spheroids in a sessile drop (SD) format (Figure 1). Benefitting from the high-aspect-ratio of recessed structures and nonwettability of the device, cell suspensions can be spontaneously partitioned into an array of superhydrophobic (SH) microwells under the combined action of surface tension and gravity, and furthermore, all partitioned droplets are stably confined in In this work, an innovative sessile drop (SD) strategy for facile, robust, and long-term cell spheroid cultures is proposed. Unlike the conventional hanging drop (HD) method, this strategy employs a superhydrophobic perforated microwell plate (SHPMP) to spatially confine arrays of cell suspension droplets for 3D spheroid cultures....

Digital polymerase chain reaction (dPCR) is emerging as a powerful method for nucleic acid detection due to its unprecedented sensitivity and precision. However, most current dPCR platforms are inherently limited by their low multiplexing ability due to primer-pair cross interactions and spectral overlap of available fluorophores. Here, we present a novel and robust method for multiplexing dPCR that is free from primer dimerization and fluorescence channel number limitation, enabling highly precise and multiplexed detection of nucleic acid targets. By prestoring target-specific primers and probes in different storage chambers, the method physically separates reactions and thus avoids the primer-pair cross interactions and spectral overlap of different fluorescent probes that usually occur within a single-tube reaction. Furthermore, a dissolvable delay valve (DDV) is embedded between each pair of the reagent prestorage chamber and reaction microwell array. Such a DDV configuration allows full reconstitution of the prestored reagents and then generates a uniform concentration distribution of the reconstituted reagents across the entire reaction microwell array, which is favorable for achieving reliable and robust multiplex dPCR assays. We demonstrated the feasibility of this method by performing an eight-plex dPCR assay targeting the seven most common point mutations in Kirsten rat sarcoma viral oncogene homologue (KRAS) and a reference sequence (wild-type KRAS allele).