Gene expression and transcript profiling involves determining the pattern of genes expressed at the transcriptional level under specific circumstances by measuring the expression of thousands of genes simultaneously. This allows one to understand cellular function. Common techniques for profiling include DNA microarrays, RNA sequencing, and EST tags. DNA microarrays involve hybridizing cDNA or cRNA samples to probes on a chip to determine relative abundance of sequences. RNA sequencing uses next-generation sequencing to reveal presence and quantity of RNA in a sample.

1. Gene expression and Transcript
profiling
priyanka Mudad
M.Sc. Biochemistry
central university of Haryana

2.  next step after sequencing a genome
 determination of the pattern of genes expressed, at the level
of transcription, under specific circumstances
 measurement of the activity (the expression) of thousands of
genes at once, to create a global picture of cellular function.
 Sequence based techniques, like RNA-Seq, provide
information on the sequences of genes in addition to their
expression level.
 expression profile allows one to deduce a cell's type, state,
environment, and so forth.

3. Methods
for
profiling
DNA
microarray
RNA Seq
EST Tags

4.  DNA chip or biochip
 collection of microscopic DNA spots attached to a solid surface.
 measure the expression levels of large numbers of genes simultaneously or
to genotype multiple regions of a genome.
 Each DNA spot contains picomoles (10−12 moles) of a specific DNA sequence, known
as probes (or reporters or oligos).
 These can be a short section of a gene or other DNA element that are used
to hybridize a cDNA or cRNA (also called anti-sense RNA) sample (called target) under
high-stringency conditions.
 Probe-target hybridization is usually detected and quantified by detection
of fluorophore-, silver-, or chemiluminescence-labeled targets to determine relative
abundance of nucleic acid sequences in the target.
DNA Microarray

5. Basic
mechanism of
DNA
Microarray
 Hybridization between the
cDNA reverse transcribed
from a biological sample
to a pre-designed
complementary DNA
probe arranged on a
slide, or array, is the
basis of DNA
microarrays.

7. Applications of Microarrays
Gene Discovery
Disease Diagnosis
Drug Discovery
Toxicological Research
For easing the accessibility to this data, the National Center
for Biotechnology Information (NCBI) has formulated
the Gene Expression Omnibus or GEO. It is a data
repository facility which includes data on gene expression
from varied sources.

8. Limitations of DNA Microarray
 cross-hybridization artifacts,
 poor quantification of lowly and highly expressed
genes
needing to know the sequence a priori

9. A/C whole transcriptome shotgun sequencing

10. RNA-Seq
 It uses next-generation sequencing to reveal the presence and quantity of RNA in a biological
sample at a given moment.
 RNA-Seq is used to analyze the continuously changing cellular transcriptome. alternative
gene spliced transcripts, post-transcriptional modifications, gene fusion, mutations/SNPs and
changes in gene expression over time, or differences in gene expression in different groups or
treatments.
 Fragmented cDNA is sequenced, the resulting sequences (“reads”) are mapped and
compared to known genomes or transcriptomes.
 In addition to mRNA transcripts, RNA-Seq can look at different populations of RNA to include
total RNA, small RNA, such as miRNA, tRNA, and ribosomal profiling.
 Three most widely used NGS platforms for RNA-seq are SOLiD and Ion Torrent, both
marketed by ThermoFisher, and Illumina’s HiSeq.

11. • Get your sample
• Lyse the cells and extract
RNA
• Convert the RNA to cDNA
• The cDNA pool get
sequenced
RNA Seq Protocol

12. Steps in RNA Seq
Library
preparation
Deep
Sequencing
Data
Analysis

13. description smallmiRNA Degradome-
seq
Digital gene
expression
Poly[A]-RNA
Seq
Total RNA seq
Library
preparation-
RNA selection
Size selection-
small RNA
Poly[A] tail
selection-
miRNA
Poly[A] tail
selection-
mRNA
Poly[A] tail
selection-
mRNA
rRNA depletion
-
mRNA+IncRNA
Sequencing
cycles
50 50 50 2×100 2×100
Single vs.
paired end
Single end Single end Single end Paired end Paired end
Data analysis discovery
expression
annotation
miRNA target
ID degradation
plots
Expression
annotation
discovery
expression
annotation
discovery
expression
annotation

17. Basic steps