Introduction high-throughput RNA sequencing over different developmental periods and

Introduction

 

Mammalian cerebrum
contain a vastly extensive variety of cells including neurons and glial cells,
which yet there has not been a reliable classification method for them. Previous
research have suggested morphological, electrophysiological and biochemical
characteristics, which could be used for their classification, however each of
them might have insufficiencies based on different types of cells and their
shared features. Therefore, there is an urgent need to unravel the differences
of central nervous system (CNS) neurons to categorize them; this has been
accomplished by using in-situ hybridization and transgene profiling (http://dx.doi.org/10.1016/j.celrep.2014.07.016). The
aim of this study is to discriminate pyramidal neurons in neocortex by using high-throughput
RNA sequencing over different developmental periods and spatial position based
on their molecular signatures.

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A high throughput RNA
sequencing provides researchers with efficient details about quantified gene
expression in addition to unraveling many unknown transcripts, alternative
splicing occurrence, and deletion of allele-specific expression. (https://dx.doi.org/10.1101%2Fpdb.top084970 abstract). In the past time, however, low
throughput sequencing methods were being used, such as northern blots and
quantitative real-time PCR, which are only capable of single transcript measurements.
Nowadays, genome-wide quantifications of gene expression, known as
transcriptomics, has  provided us with a
tremendously broad spectrum of knowledge about molecular signatures, ergo an
expeditious progress in cellular classifications.

Regarding the high
complexity of the cellular stages during developmental periods and the
dynamicity of their features, three sub-types of pyramidal neurons have been
selected for whole-transcriptome analyses by massively parallel RNA sequencing.
The targeted cells for this study were Subcerebral projection neuron (ScPN), callosal
projection neuron (CPN) and corticothalamic projection neuron (CThPN) based on
their fundamental role in central nervous system including their spatial
placements, clinical relevance and the tremendously important cognitive roles.
( http://dx.doi.org/10.1016/j.neuron.2014.12.024)

In this study, RNA
samples were obtained and purified from specified neurons. Fluorescent activated
cell sorting (FACS) was used for immunodetection of the nuclear markers. This
method is a valuable approach to determine molecular features of a cell,
however this might arise some complications, such as the vulnerability of RNA
molecules to degradation, or the limitation of fluorescent tags that could be
used at the same time causing to neglecting of neural details. Hence, to
confirm the quality and quantity of the RNA sample, RNA integrity number (RIN)
has been used as an index. (RIN  is indicative of a suitable RNA sample).

Three main
transcription markers have been selected as a discriminatory index, which are
Satb2, Bcl11b, and Tle4. The markers were selected based on their adequate
presence in all developmental courses of every neuron in the study. Their
adequate presence in the cells were examined using qRT-PCR to make sure that
these markers are present in the targeted neurons in addition to a
clarification that the screened cells are not glial cells. Fig.12