“We would like to argue that the most accurate method for transcriptome analysis is sequencing,” notes Kai Lao, Ph.D., principal scientist, genetic systems, Life Technologies. “Recent advances in high-throughput sequencing made it possible to analyze single-cell transcriptomes at high resolution.”
Life Technologies used the SOLiD next-generation sequencing technology for deep sequencing of transcriptomes of mouse oocytes and stem cells. Single-cell RNA-Seq overcomes the typical limitations of microarray analysis while achieving greater accuracy.
To illustrate the accuracy of the method, researchers used genetically modified oocytes lacking only one exon of a single-copy gene (Dicer). Reads that map to exon 23 were entirely absent, whereas the reads from neighboring exons were intact. Overall, the method detects 94% of all expressed genes in a single cell.
The assay can also be used to discover new transcripts and alternative splicing isoforms. It also facilitates quantitative estimates of RNA abundance by the frequency with which the sequence occurs in the RNA-Seq reads. Recent experiments have already generated the insights into possible pathways for derivation of embryonic stem cells (ESCs) from the inner cell mass of blastocysts.
While the cells of the blastocyst follow a strict developmental program, the stem cells are self-renewing and pluripotent. Sequencing of RNA of single cells at different points in the transition from ICM to ESCs demonstrated multiple and profound changes in the transcriptome affecting epigenetic regulators, microRNAs, and pluripotency gene clusters.
Tracking mRNA in a Living Cell
All cells including stem cells and progenitor cells undergo changes over time, which is reflected in their mRNA and protein profiles. Techniques that allow multiple measurements in the same live cell over a period of time are vital for understanding the influence of individual cells on the cellular systems.
Fluorescent imaging has become sensitive enough to measure levels and location of tagged proteins inside a living cell. However, tracking individual mRNA had remained a challenge until the group from Bar-Ilan University, led by Yaron Shav-Tal, Ph.D., developed a real-time detection system for mRNA.
The technology is based on creating a modified 3´ end of a target gene by incorporating multiple repeats of a binding site for MS2, an RNA-binding protein. In turn, MS2 protein is coupled with the green fluorescent protein (GFP). One mRNA transcript binds to about 30 MS2-GFP proteins generating an easily detectable fluorescent signal.
“This was the first instance of measuring transcription of a single gene in vivo in human cells. Now we are able to detect mRNA translocation from the nucleus to cytoplasm. The signal is visible at 3–5 mRNAs per gene,” says Dr. Shav-Tal.
The first few experiments have already shed light on such important questions as the activity of viral promotors in comparison to native promotors. While the native promoter shut down for about 20 minutes every 200 minutes, the viral promoter remained active for hours.
In the future, this technology may provide the answers to how manipulations of transcription increase or decrease expression of disease-related genes. By introducing specific mutations, scientists will determine their influence on promoter potency and therefore gene expression.
“We will be able to research interactions between replication and transcription machinery,” continues Dr. Shav-Tal. “And how the external signal reaches specific gene promotors and turns them on or off.”
A recent development includes a knockin mouse carrying a target gene coupled with MS2 binding sites. Now every cell of the mouse carries this transcriptional reporter. This in vivo mRNA reporting system opens amazing possibilities for tissue-specific comparisons and for studying the signal transduction effects throughout the whole organism.
Infrared spectroscopy enables visualization of gross differences between individual cells based on their absorption spectra. A cell can be arrested at a particular point by formalin fixation, after which the IR spectra can be obtained within just a few minutes.
“Infrared cytology is an objective analysis of the cells and brings a unique angle to cytological examination,” comments Peter Gardner, Ph.D., director of assessment at the School of Chemical Engineering and Analytical Science, The University of Manchester. Dr. Gardner anticipates that IR spectroscopy will become an integral part of diagnostics, helping to identify and grade diseased cells in pathology specimens.
Another opportunity lies in personalized medicine. Instead of searching for specific molecular markers to predict a patient’s response to drug therapy, the same prediction can be made by using IR analysis of the person’s own cells.
Using ovarian cancer cell lines, Dr. Gardner’s group demonstrated clear spectroscopic separation between cell lines responding and not responding to treatment with the cytotoxic drug cisplatin. Moreover, when treated with a new experimental compound, KF101, the cells presented yet another unique spectral signature. This observation indicated that KF101 acts by a mechanism different from that of cisplatin.
“I see another opportunity for IR spectroscopy in studies of stem cells,” continues Dr. Gardner. “Stem cell differentiation induces changes in the spectrum. If stem cell implantation has to happen at a certain differentiation stage, the IR spectral data would provide a robust measure of whether the cells have reached that stage.”
Dr. Gardner says that he and his colleagues have resolved a long-standing issue of spectral distortion. Mid-infrared radiation scatters strongly from the organelles distorting the absorption spectrum. The scientists modified the existing Mie scattering theory, which describes scattering of light from the objects of a size comparable to the illuminating wavelength.
A new mathematical algorithm was able to correct the spectral distortion. This methodology was recently applied to identification of stem cells among a side population of tumor cells. This population is thought to be enriched with stem cells, but their low counts prevent further fractionation and characterization. FTIR spectroscopy was able to record the fingerprints of individual cells, confirming their biochemical differences.