We describe a technique for profiling microRNAs in early mouse embryos. This protocol overcomes the challenge of low cell input and small RNA enrichment. This assay can be used to analyze changes in miRNA expression over time in different cell lineages of the early mouse embryo.
MicroRNAs (miRNAs) are important for the complex regulation of cell fate decisions and developmental timing. In vivo studies of the contribution of miRNAs during early development are technically challenging due to the limiting cell number. Moreover, many approaches require a miRNA of interest to be defined in assays such as northern blotting, microarray, and qPCR. Therefore, the expression of many miRNAs and their isoforms have not been studied during early development. Here, we demonstrate a protocol for small RNA sequencing of sorted cells from early mouse embryos to enable relatively unbiased profiling of miRNAs in early populations of neural crest cells. We overcome the challenges of low cell input and size selection during library preparation using amplification and gel-based purification. We identify embryonic age as a variable accounting for variation between replicates and stage-matched mouse embryos must be used to accurately profile miRNAs in biological replicates. Our results suggest that this method can be broadly applied to profile the expression of miRNAs from other lineages of cells. In summary, this protocol can be used to study how miRNAs regulate developmental programs in different cell lineages of the early mouse embryo.
A central question of developmental biology is how a single undifferentiated cell can give rise to an entire organism with numerous complex cell types. During embryogenesis, the developmental potential of cells becomes progressively restricted as the organism develops. One example is the neural crest lineage, which progressively differentiates from a multipotent cell population into various terminal derivatives, such as peripheral neurons, glia, cranial bone, and cartilage. Neural crest cells are specified from the ectoderm during gastrulation and then undergo an epithelial to mesenchymal transition and migrate through the embryo to discrete locations throughout the body where they will terminally differentiate1. Decades of work have uncovered a transcriptional gene regulatory network, but far less is known about the mechanisms of post-transcriptional regulation that control the timing of neural crest development.
Previous work suggests that microRNAs (miRNAs) repress gene expression for proper developmental timing and cell fate decisions2,3,4,5,6. Studies of miRNAs in neural crest development have largely focused on later stages of craniofacial development. For example, miR-17~92 and miR-140 are critical for palatogenesis during craniofacial development in mouse and zebrafish, respectively7,8. The contribution of miRNAs to the earliest neural crest fate decisions of the embryo has not been thoroughly investigated. Studies of miRNAs in early fate decisions have been limited by technical challenges such as the low cell number present in early embryos.
MiRNAs have been profiled in vitro from cell lines using embryoid bodies at different stages of differentiation to model early mouse development9. The investigation of small RNAs in vivo during early mammalian development has been relatively limited. Previous methods to profile miRNAs have led to bias as a known sequence is used to analyze expression of a specific miRNA in methods such as qPCR, microarrays, and northern blots10. Next generation sequencing and ever improving molecular tools now allow for relatively unbiased analysis of miRNA expression to study their contribution to early mammalian development and cell fate decisions.
Here, we report a technique to harvest and sequence small RNAs expressed in neural crest cells from early mouse embryos spanning gastrulation (E7.5) to the beginning of organogenesis (E9.5). This technique is straightforward and combines lineage tracing, cell sorting, and gel-based size selection to prepare small RNA sequencing libraries from a minimal number of cells for next generation sequencing. We highlight the importance for strict somite stage matching of embryos to resolve 6-hour time intervals to obtain a comprehensive view of miRNAs during the rapid changes of early development. This method can be widely applied to genetic and developmental studies and avoids the pooling of embryos. We describe a way to overcome challenges of current methods such as miRNA enrichment using gel-based purification, library quantification, and minimizing bias introduced from PCR. This method has been used to identify miRNA expression patterns over time to study how miRNAs control developmental timing in the neural crest lineage of mouse embryos.
Developmental processes can proceed rapidly, and cells are undergoing many sequential specifications such that to capture a comprehensive view of the miRNAs contributing to early fate decisions, more specific staging is needed than the widely used half-day increment. A recent study has performed RNA sequencing from Theiler stage 12 embryos which range from having 3 to 6 somites11. We find that during this period of time, the neural crest cells are specified (3 somites of age), delaminate (4 somites of age), and migrate (5-6 somites of age). We also find that besides the Cre-driver used to lineage trace cell populations, age is the largest source of variation between biological samples, and only somite matched embryos should be considered as replicates. This should also be taken into consideration when comparing transgenic embryos to wildtype controls.
Previous methods to profile miRNAs during early development have used between 10-100 ng of RNA input for small RNA sequencing library preparation and have pooled multiple embryos into one sample13,14. We demonstrate RNA isolation and library preparation from a single E7.5 embryo or from sorted neural crest cells at E8.5 and E9.5 using approximately 100 ng of total RNA as input. When dissociating embryos for sorting, one should take care to watch the dissociation under a microscope and quench the reaction to observe when single cells are obtained. We find that dissociation of the cranial region of E8.5-E9.5 embryos is almost instant with gentle manual pipetting as described in the protocol. For larger tissues and increasingly older embryos, dissociation time may be longer depending on the portion of the embryo being dissociated. For E7.5-E9.5 embryos, clumps of cells are easily visible under the microscope and the dissociation should continue until no more clumps are visible. Single cells are visible in solution if you adjust the focus through the solution in your well anywhere from 5-10x. Previous methods sort cells directly into lysis buffer for RNA sequencing to prep bulk RNA from a low number of cells14. Here we sort directly into RNA extraction lysis solution so that RNA can be isolated before the start of library preparation. Use of mini columns with 11 µL elution volume allowed for a high enough RNA concentration such that a single RNA prep could be split between small RNA and bulk RNA sequencing.
One current limitation of most small RNA sequencing methods is the PCR amplification of converted cDNA. Our method does not overcome this limitation, but we were able to minimize the number of PCR cycles from the 25-maximum recommended down to 16 cycles. This reduction in amplification decreases artificial amplification bias introduced by PCR. Another source of bias is the ligation of adapters, where it is known that specific sequences located at the ends of adapters and miRNAs can ligate together with greater efficiency than other sequences. To avoid this, the adapters used in this protocol have 4 random bases incorporated at the end of each adapter to prevent bias in ligation reactions. Additionally, another common issue is the amount of adapter dimers that form when the RNA input is low. The library preparation kit does include steps to reduce adapter dimer formation such as adapter inactivation and bead cleanups to remove excess adapters after each ligation. We also diluted the 3’ and 5’ 4N adapters by ¼ to reduce the amount of adapter dimer that can form. We found that when not diluted, the 130 bp band intensity increases making it difficult to distinguish from the 150 bp band containing the desired small RNA libraries on a gel.
Another current challenge of preparing sequencing libraries is the accurate quantification of product prior to sequencing. We have found that different methods give varying results on the same library. We suggest that the researchers use multiple methods of quantification to get an accurate estimation of concentration.
This protocol can be widely applied to genetic, developmental studies, or other applications where RNA is being harvested from a low number of cells. This approach simplifies temporal studies by avoiding the pooling of embryos and can easily be applied to both non-sorted and sorted cells.
The authors have nothing to disclose.
This project was supported by Andrew McDonough B+ Foundation and the NIH (R01-HD099252, R01-HD098131. R.J.P. is a CPRIT Scholar in Cancer Research (RR150106) and V Scholar in Cancer Research (V Foundation). The authors would also like to acknowledge the Cytometry and Cell Sorting Core at BCM for providing equipment necessary for this project.
#5 Forceps Fine Science Tools | Fisher Scientific | NC9277114 | |
0.4% Trypan blue | ThermoFisher | 15250061 | |
10 cm Petri dishes | Fisher Scientific | 07-202-011 | |
2-Propanol | Miilapore Sigma | I9516-500ML | |
5 % Criterion TBE Polyacrylamide Gel, 12+2 well, 45 µL | Bio-Rad | 3450047 | |
5200 Fragment Analyzer | Agilent | M5310AA | |
96 well PCR plates high profile semi skirted clear/clear | Bio-Rad | HSS9601 | |
BD FACSAria II | bdbiosciences | ||
Bovine Serum Albumin, fraction V | Fisher Scientific | BP1600100 | |
Dulbecco's Phosphate-Buffered Saline (DPBS) | Gendepot | CA008-300 | |
Eppendorf tubes | Fisher Scientific | 05-408-129 | |
Ethanol Pure, 200 proof anhydrous | Sigma Aldrich | E7023-500ml | |
FC-404-2001 | Illumina | FC-404-2001 | |
HS NGS Fragment kit | Agilent | DNF-474-0500 | |
HS RNA Kit | Agilent | DNF-472-0500 | |
miRNeasy Mini Kit (50) | Qiagen | 217004 | |
NEXTflex Small RNA-Seq Kit v3 (48 barcodes) | Fisher Scientific | NC1289113 | |
NextSeq 500/550 Mid Output Kit v2 (150 cycles) | Illumina | FC-404-2001 | |
NGS Fragment kit | Agilent | DNF-473-1000 | |
Papain | Worthington | LK003176 | resuspend in 5 mL of Ca/Mg free PBS for a final concentration of 27 U/mL |
SYBR Gold nucleic acid gel stain | ThermoFisher | S11494 | |
Trizol LS | ThermoFisher | 10296028 | |
UVP Benchtop UV Transilluminators: Single UV | Fisher Scientific | UVP95044701 | |
Vannas Scissors Straight Fine Science Tools #91500-09 | Fisher Scientific | NC9609583 |