trajectory of single cells offers an approach to profile the molecular
changes that occur in each cell undergoing the EMT over time.
This approach therefore, has the potential of eliminating much of
the noise introduced either by asynchrony of the EMT and/or
inclusion of noninvolved cells, and the reward is provision of a
temporal profile of molecular change.
It should be noted, however, that scRNA-seq is not the perfect
solution. Because of the small amount of RNA obtained from each
cell, amplification is necessary before sequencing. This and other
limitations means that some rare RNA species are less likely to be
included in the database than in bulk RNA-seq approaches. Nevertheless, the advances in scRNA-seq approaches provide the investigator with a valuable tool to penetrate EMT mechanisms to a level
that heretofore has been unreachable.
2 The Single Cell RNA-Sequencing Approach, a Justification
Next generation sequencing (NGS) platforms increasingly allow
in-depth analyses of gene expression and genetic interactions in
many biological systems. The approaches allow the investigator
unprecedented access to biological questions. The methodology
begins with sample preparation, includes library production,
sequencing, and data analysis. The latter is most important as
software continues to be developed to enable the investigator to
gain ever more detail about the biological process in question. As
part of the description, the caveats and limitations of these technologies will be discussed. The focus will be on approaches that
advance RNA-sequencing technologies and their application to
understanding EMTs.
Two methods of RNA-sequencing are currently utilized, single
cell RNA-sequencing (scRNA-seq) and bulk RNA-sequencing
(RNA-seq). They each have their own individual advantages and
disadvantages and are useful for addressing different biological
questions. Bulk (whole-tissue) RNA-sequencing has many applications for research including comparative gene expression analyses
between samples of various conditions, differential gene expression,
identification of mRNA splice variants and small or long noncoding
RNAs. RNA material collected from whole-tissue samples requires
less or no amplification relative to scRNA-seq and the sample can be
more deeply sequenced than that obtained from a single cell. Bulk
RNA-seq is also easier: obtaining single cell suspensions from fixed
or frozen tissue is nontrivial and may be very difficult for some
samples. Thus, bulk RNA-sequencing is a good option in many
applications. However, bulk RNA-seq is not as informative for
identifying transcriptional differences within heterogeneous cell
populations such as in developing and complex tissues because
bulk RNA-seq measures the expression level of transcripts across a
Methods for in Vivo EMT at Single Cell Resolution
305
changes that occur in each cell undergoing the EMT over time.
This approach therefore, has the potential of eliminating much of
the noise introduced either by asynchrony of the EMT and/or
inclusion of noninvolved cells, and the reward is provision of a
temporal profile of molecular change.
It should be noted, however, that scRNA-seq is not the perfect
solution. Because of the small amount of RNA obtained from each
cell, amplification is necessary before sequencing. This and other
limitations means that some rare RNA species are less likely to be
included in the database than in bulk RNA-seq approaches. Nevertheless, the advances in scRNA-seq approaches provide the investigator with a valuable tool to penetrate EMT mechanisms to a level
that heretofore has been unreachable.
2 The Single Cell RNA-Sequencing Approach, a Justification
Next generation sequencing (NGS) platforms increasingly allow
in-depth analyses of gene expression and genetic interactions in
many biological systems. The approaches allow the investigator
unprecedented access to biological questions. The methodology
begins with sample preparation, includes library production,
sequencing, and data analysis. The latter is most important as
software continues to be developed to enable the investigator to
gain ever more detail about the biological process in question. As
part of the description, the caveats and limitations of these technologies will be discussed. The focus will be on approaches that
advance RNA-sequencing technologies and their application to
understanding EMTs.
Two methods of RNA-sequencing are currently utilized, single
cell RNA-sequencing (scRNA-seq) and bulk RNA-sequencing
(RNA-seq). They each have their own individual advantages and
disadvantages and are useful for addressing different biological
questions. Bulk (whole-tissue) RNA-sequencing has many applications for research including comparative gene expression analyses
between samples of various conditions, differential gene expression,
identification of mRNA splice variants and small or long noncoding
RNAs. RNA material collected from whole-tissue samples requires
less or no amplification relative to scRNA-seq and the sample can be
more deeply sequenced than that obtained from a single cell. Bulk
RNA-seq is also easier: obtaining single cell suspensions from fixed
or frozen tissue is nontrivial and may be very difficult for some
samples. Thus, bulk RNA-sequencing is a good option in many
applications. However, bulk RNA-seq is not as informative for
identifying transcriptional differences within heterogeneous cell
populations such as in developing and complex tissues because
bulk RNA-seq measures the expression level of transcripts across a
Methods for in Vivo EMT at Single Cell Resolution
305