differential gene profiling methods

1. Differential gene profiling
methods
Sonam Yadav
M.Sc. Botany 4th Sem.

2. Differential gene profiling
• Gene expression profiling is the measurement of
the activity (the expression) of thousands of
genes at once, to create a global picture of
cellular function.
• These profiles can distinguish between cells that
are actively dividing, or show how the cells react
to a particular treatment. Many experiments of
this sort measure an entire genome
simultaneously ; every gene that is present in a
particular cell.

3. Differential gene expression
• Through the process of differential gene
expression, the activation of different
genes within a cell that define its purpose,
each cell expresses only those genes which it
needs. However, the extra genes are not
destroyed, but continue to be stored within
the nucleus of the cell.

4. Methods of gene expression

5. Why is differential gene profiling
important?
• Gene expression profiling measures which
genes are being expressed in a cell at any
given moment
• This method can measure thousands of genes
at a time; some experiments can measure the
entire genome at once.

6. Eg.-Differential gene expression in leaf tissues between mutant
and wild-type genotypes response to late leaf spot in peanut
(Arachis hypogaea L.)

8. • Analysis of the differential gene and protein
expression profile of the rolled leaf mutant of
transgenic rice (Oryza sativa L.)

9. TYPES
1. Differential display
2. Subtractive hybridization

10. DIFFERENTIAL DISPLAY
• Differential display is a powerful technique for
detecting and quantitating changes in gene
expression patterns between differently treated
cells.
• Fragments of those genes which are induced or
suppressed can be identified and isolated for
further analysis, with no prior knowledge of the
sequences involved. The technique is PCR based,
and yields results in only 1 - 3 days.
• DDRT-PCR was developed by Liang and Pardee.

12. •Differential display (also referred to as DDRT-PCR or DD-
PCR) is a laboratory technique that allows a researcher to
compare and identify changes in gene expression at
the mRNA level between two or more eukaryotic cell
samples.
•It was the most commonly used method to compare
expression profiles of two eukaryotic cell samples in the
1990s.
• By 2000, differential display was superseded by DNA
microarray approaches.

13. POTENTIAL APPLICATIONS
• DDRT-PCR has been used to answer many
biological questions in mammalian and other
biological systems.
• RT-PCR to examine the expression of
each FRO gene in different tissues of
Arabidopsis ‘s root and in response to iron
and copper limitation.
• (FRO- Ferric reductase oxidase)

14. • This method is also used in the identification
of differentially expressed genes in developing
cotton fibre.
• The multidimensional applications of DDRT
PCR in study the differentially expressed gene
under biotic and abiotic stress especially the
salinity stress.
Eg. In Rice (Oryza sativa) -Expression
of SAMDC1 gene positively correlates with salt
tolerance in rice.

15. SUBTRACTIVE HYBRIDIZATION
• Subtractive hybridization is a technique for
identifying and characterizing differences
between two populations of nucleic acids.
• It detects differences between the RNA in
different cells ,tissues,organisms,or sexes under
normal conditions,or during different growth
phases,after various treatments (i.e, Hormone
application,heat shock) or in diseased(or mutant)
versus healthy(or wild-type) cells.

16. • The two population are hybridized with a
driver to tester ratio of at least 10:1.
• Because of the large excess of driver
molecules, tester sequences are more likely to
from driver-tester hybrids than double
standard tester.
• Only the sequences in common between the
tester and the driver hybridize , however,
leaving the remaining tester sequences either
single standard or forming tester-tester
sequences.

17. • The driver-tester ,double-standard and any
single standard driver molecules are
subsequently removed(the “subtractive”step,
leaving only tester molecules enriched for
sequences not found in the driver.
T D
T TT TD DD D
× × × ×

18. PROCESS
Choosing material for isolating tester and driver
nucleic acid.
producing tester and driver.
hybridizing them
Removing driver-tester hybrids and excess
driver(subtraction)
Isolating of the complete seq. of remaining
target nucleic acid.

20. Applications
• simplified, fast and reliable method for generating
subtracted probes or subtracted cDNA libraries.
• Only small amounts of sample/mRNA are required
• Magnetic handling minimizes losses at each step
• Specific mRNAs are highly enriched
• Magnetic handling enables simple and rapid buffer
changes to optimize conditions for hybridization and
specific enzymatic reactions.

21. REFRENCES
• www.google.com,wikipedia.
• Subrahmanyam P, Rao VR, McDonald D, Moss JP, Gibbons RW. Origins of
resistances to rust and late leaf spot in peanut (Arachis hypogaea,
Fabaceae). Economic Botany. 1989; 43(4):444–455.
• pandey A; Mann, M (15 June 2000). "Proteomics to study genes and
genomes". Nature. 405 (6788): 837–46. .
• Reece, Richard J. (2004). Analysis of genes and genomes. Chichester:
Wiley. ISBN 978-0-470-84380-2
• .
• Galbraith, E. A.; Antonopoulos, D. A.; White, B. A. (2004). "Suppressive
subtractive hybridization as a tool for identifying genetic diversity in an
environmental metagenome: The rumen as a model". Environmental
Microbiology. 6 (9): 928–937. doi:10.1111/j.1462-
2920.2004.00575.x. PMID 15305918.
•