Cell fixation and preservation for droplet-based single-cell transcriptomics

In the current progress in the droplet-based microfluidics that allows the transcriptions characterizing of thousands of individual cell in a highly quantified, collateral, and penurious way. The crucial stage which is often a restrictive step is the composition of cells in an unruffled state not being modified by stress or age. In addition to this, the other strenuous work is to gather rare cells over many days or specimen prepared at various places or times.

Background:

A tissue is comprised of various characteristic cell types, where each of them has a different biological state. Instead of getting to study the entire gene expression of the tissue in the common, it has been identified that transcriptional profiling at a single cell resolution gives the exact and entire biological function of it.

In the droplet sequencing’ method, cells are individually concentrated in nanoliter-sized droplets along with a single drip in a microfluidic device. This is followed by a polymerase chain reaction (PCR), unique molecular identifiers (UMIs), and then polyT sequence. Later a reverse transcription of mRNA is carried to analyze which of the mRNAs originated from the same cell. In spite of having other methods of performing the sequencing, mRNA- seq is highly preferred due to its ability to not limit the input variables during the preparation process. The main aspect to be considered is to obtain valuable and credible information of single-cell suspension that depicts the transcriptional expression is every cell in its own environment or natural state through the experiment. The most challenging part of it is where the possibility for changes in the transcriptome is possible and identifying it is significant. Another possibility is where the tissues might get combined in the experimental process subjected to certain restrictions.

Method:

The chemical fixation method is adopted to rectify these issues. Aldehydes, methanol, and ethanol are coagulating fixative that chemically does not alter the nucleic acids. The methanol fixation enabled to equilibrate and maintain segregated cells for weeks with compensating on the single-cell RNA series data. In this method, the cells were mostly monitored under low-temperature level to avoid cell loss and hence was kept under a cold environment at all means. The methanol mixed cells were placed on ice for a minimum of 15 minutes and then was stoked at -80°C for more than months. It is later moved to ice fixation from -80°c to 4°C but maintained to be kept cold throughout the process.

By using blends of fixed, cultured human and mouse cells, it was first identified that single transcriptomes could be positively allowed to any one of the two specimens. Single-cell gone expression from live and secured samples corresponded well with heavy mRNA- sequencing data. Then when applied with methanol fixation to transcriptionally figured primary cells from segregated, compound tissues. The cells with low RNA component from Drosophila embryos, also from mouse hindbrain and cerebellum cells sorted out by activating fluorescence- cell sorting, a successful fixation was analyzed in storage, and a single droplet of RNA sequencing. We could notice the multiplication of cell population, involving neuronal categories. The droplet sequencing toolkit had been additionally implied to impose new gene annotations within the positioned reads and to rectify the possible complexity (synthetic errors) as described above. Each of the barcodes associated with the single-cell transcriptome was determined by the number of reads observed in each cell.

Conclusion: Preserving and faxing the cells at the initial step drives them away from becoming biased and from any other technical differences, also from unexpected changes in the cell (ageing, etc) through the experimental process. Also, cell fixation eases down the logistic collaboration of high-scale experiments. For instance, obtaining the details on rare cells cannot be found through a simple process or any clinical method, but requires high specification/ extensive methods to pull out the accurate data. We hope that the presence of a simple cell fixation method might show up varied new opportunities in the distinct biological context to evaluate transcriptional dynamics at single-cell resolution.

Anacyte Laboratories is dedicated to facilitate and improve molecular analyses of biomolecules. Anacyte develops state of the art systems for bio-molecule analyses in the post-genomic era of sciences, focusing on the understanding of genetic circuits, which enable sophisticated regulatory networks to specify cells´ fate, development, and function.

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