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Microarray Introduction

MicroArray and it's procedure

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3 views

Microarray Introduction

MicroArray and it's procedure

Uploaded by

zohasaleem14.02
Copyright
© © All Rights Reserved
Available Formats
Download as PPTX, PDF, TXT or read online on Scribd
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AN INTRODUCTION TO GENE EXPRESSION

ANALYSIS BY MICROARRAY TECHNIQUE

DR SAJED ALI
INCHARGE PROGRAM BIOTECH/BIOCHEM
VOCABULARY

• Gene: hereditary DNA sequence at a specific


location on chromosome

• Genetics: study of heredity & variation in


organisms

• Genome: an organism’s total genetic content (full


DNA sequence)

• Genomics: study of organisms in terms of their


genome
VOCABULARY

• Bioinformatics: the collection, organization, &


analysis of large scale, complex biological data

• Statistical bioinformatics: the application of


statistical approaches to bioinformatics,
especially in identifying significant changes (in
sequences, expression patterns, etc.) that are
biologically relevant
THE BIOLOGY BACKGROUND OF
MICROARRAY

 The central dogma of molecular biology


 DNA
 RNA
 Monitoring the expression of genes
CENTRAL DOGMA OF
MOLECULAR BIOLOGY
DNA replication

DNA Transcription

RNA Translation

Protein

Reverse
transcription
DEOXYRIBONUCLEIC ACID
(DNA)
The double helix
• read from 5’ to 3’
• antiparallel: one strand has
direction opposite to its
complement’s
Nucleotide
• A, T, G, C
Base pair
• A – T (2 H-bonds)
• G – C (3 H-bonds)
Oligonucleotide
• short DNA (tens of nucleotides)

(http://www.nhgri.nih.gov)
HYDROGEN BOND MAKES DNA
BINDING SPECIFICALLY
Hydrogen bond

’5

’3

’5

’3
RIBONUCLEIC ACID
(RNA)

• RNA is much more abundant than DNA

• There are several important differences between RNA


and DNA:
1. The pentose sugar in RNA is ribose, in DNA it’s deoxyribose.
2. In RNA, uracil replaces the base thymine (U pairs with A).
3. RNA is single stranded while DNA is double stranded.
4. RNA molecules are much smaller than DNA molecules.
TYPES OF RNA

There are three main types of RNA:


1.Ribosomal (rRNA)
2.Messenger (mRNA)
3.Transfer (tRNA)
REVERSE TRANSCRIPTION
replication
transcription translation

DNA RNA Protein

Reverse Transcription

• Gene is expressed by transcribing DNA into single-


stranded mRNA
• By reverse transcriptase, we can convert RNA into
complementary DNA (cDNA)
MESSENGER RNA REPRESENT
GENE FUNCTION
• When measure the level of a mRNA, we are monitoring
the activity of a gene

• Thus, if we can understand all the level of mRNAs, we


can study the expression of whole genome

• Microarray takes the advantage of getting over 10000 of


blotting data in a single experiment, which makes
monitoring the genome activity possible
MICROARRAY

• Microarray refers to types of massively parallel biological


assays where many tests are done simultaneously

• The word “Microarray” has become a general term, there


are many types now
DNA microarrays
Oligonucleotide microarrays
Protein microarrays
Transfection microarrays
Tissue microarray
OLIGONUCLEOTIDE MICROARRAY
AN OVERVIEW

• The abundance and constancy of proteins in a cell determine the


functions of it. Thus, the function or activity of a gene is reflected by the
synthesis of mRNA (transcription) or protein (translation)

• Microarrays represent a major technology in the field of molecular


biology and medicine

• Oligonucleotide microarray is also known as Affymetrix GeneChip


Array or one color array

• It has become a powerful technique in measuring gene expression


levels (at a transcriptional level) in order to improve diseases diagnosis
as well as to create new effective treatment regimens
OLIGONUCLEOTIDE MICROARRAY
THE IMPORTANCE

Understand the transcription level of gene(s)


under different conditions such as:

Cell types (brain vs. liver)


Developmental (fetal vs. adult)
Response to stimulus (rich vs. poor media)
Gene activity (wild type vs. mutant)
Disease states (healthy vs. diseased)
WHAT CAN WE LEARN BY
ANALYZING COMPLEX PATTERNS OF
GENE EXPRESSION?
1. Classifications: for diagnosis, prediction…
• Cell-type
• Stage-specific
• Disease-related
• Treatment-related patterns of gene expression

2. Gene Networks/Pathways:
• Functional roles of genes in cellular processes?
• Gene regulation and gene interactions
OLIGONUCLEOTIDE MICROARRAY
ARRAY DESIGN
• Each array is composed of thousands of DNA oligonucleotides spots
(probes) that are factory designed and synthesized attached to a solid
support

Raw image
1.28cm

50um

,half perfectly match mRNA (PM)


half have one mismatch (MM)
Raw gene expression is intensity
difference: PM - MM
OLIGONUCLEOTIDE MICROARRAY
(C) ARRAY DESIGN
• Usually for each gene, (11-25) pairs of probes are synthesized on the chip
from the 3’ end of each transcript

• Each pair of probes have two oligonucleotides:


1. Perfect match probe (PM) which has complete complementary sequence to the
sequence of the reference
2. Mismatch probe (MM) has a single mismatch to the target sequence, centred in
the middle of the nucleotide, usually nucleotide number 13 has been changed

• The number of PM and MM probes used varied from array to another


depending on specific performance criteria for each assay

• In general, (MM) probes are used to minimize unspecific binding during


hybridization (cross-hybridization)
http://www.affymetrix.com/technology/ge_analysis/index.affx
OLIGONUCLEOTIDE MICROARRAY
(D) ADVANTAGES VS. DISADVANTAGES
ADVANTAGES DISADVANTAGES

•Highly hygienic chips since these •Highly cost to design


chips are synthesized by a certain chip
photolithography process
•The need to access to
expensive specialised
•Probes design is based entirely equipment to run the
on sequencing information, there analysis
is no need for the physical
intermediates such as bacterial •Short sequence nucleotide
plasmids or PCR products, which probes may decrease the
results in a minimum chance to sensitivity of binding in
create probes mix-up comparing with DNA
Microarray
OLIGONUCLEOTIDE MICROARRAY
(E) QUALITY CONTROLS
• Many factors or criteria should be involved in each
array to ensure the ideal quality control and accuracy
of the array such as:

1. The number of sample replicates


2. RNA isolation
3. RNA integrity number (RIN)
4. Microarray hybridization controls
OLIGONUCLEOTIDE MICROARRAY
(E) QUALITY CONTROLS

1. The number of sample replicates


 It varies from one species to another depending on the source of
biological variability in the sample to be examined such as stage of
disease

2. RNA isolation (quality and the quantity of RNA sample)


 Many methods can be used to assess RNA quantity & quality: UV ratio
of 260/280 (should be around 1.8-2.1), Agarose gel electrophoresis or
on Agilent Bioanalyzer to visualize the 18s/28s ribosomal subunits
bands
 RNA quantity should be ranged from 5-10 µg to yield biotinylated
complementary RNA (cRNA) between 4-10 fold greater than the total
RNA sample used otherwise the sample could not be run on Affymetrix
GeneChip Microarray
Electropherogram of RNA samples on Agilent
2100 bioanalyzer

28s rRNA

18s rRNA
OLIGONUCLEOTIDE MICROARRAY
(E) QUALITY CONTROLS

3. RNA integrity number (RIN)


 It is a measurement of RNA integrity and degradation
ranges from 1 to 10, where 1 is the most degraded RNA
and 10 is the most intact RNA
 For Affymetrix GeneChip Microarray, RIN should be (≥6)

4. Microarray hybridization controls


 Several hybridization controls including the visualization of
the image are included to check any abnormalities in the
hybridization patterns
General metrics for overall Affymetrix GeneChip quality

Criteria Description

The scaling factor It should be around 3, but it can be acceptable as long as it is not ≥ 5

It should be around 40-50%, but it can be acceptable as long as it is > 25%. This
% Present calls
number can vary regarding to tissue type

It gives indication about the labelling step of the array and it gives information for
Sig (3’/5’) ratio
GAPDH and B-ACTIN. In general, it should be < 3

Background (BG) noise It should be <100

)BioB, BioC, BioD, Cre( It is important to describe the hybridization process. BioB is only present half of the

controls time while, BioC, BioD and Cre should always have a present call
OLIGONUCLEOTIDE MICROARRAY
(F) WORK FLOW

http://www.affymetrix.com/technology/ge_analysis/index.affx

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