Microarray and DNA chips for Transcriptome study

Amna Siddiqui, Areeba Khan, Faiq Sabih, Hammad Naveed, Hassan Fajri

BBS-6

Overview

• Transcriptome study; why needed?• Methods used• What are Microarray and DNA chips?• Difference between the two• Method used to study one or more transcriptome• Complications• Alternate method to compare two transcriptomes• Yeast transcriptome• Applications

TranscriptomeA transcriptome is the collection of all the mRNA transcripts transcribed from the DNA (genome), at any one time, in a particular cell. • 4% of the total RNA content of the cell • Specifies the composition of the proteome (concerned with coding regions that will be expressed)• Expression triggered by environmental factors • If conditions are not optimum, total expression can be switched off.

Transcriptome study• Study of the transcriptome of any organism’s cell is

also called expression profiling• By analyzing transcriptome sequence, it can be

determined when and where a gene is turned off or on in a particular cell

• Organisms may have same genes but different gene expression leading to difference in behaviours

• Thus, comparison of two transcriptomes (from different specie or different cell types) can be used to determine properties of each cell type

• Doing transcriptome study of a diseased or cancerous cell can lead to information about the gene responsible for producing abnormality (gene expression less or more)

Methods used in Transcriptome Study

Serial Analysis of Gene

Expression (SAGE)

Massively parallel

signature sequencing

(MPSS)

Microarray and DNA chips

RNA-Seq (emerging method)

Microarray and DNA chips• Microarray method used to check gene

expression• Microarrays or DNA chips use a thin glass

microscopic slide, silicon chip or nylon membrane

• Thousands of reference genes can be immobilized, spotted or synthesized in situ on a small space on these glass slides

• Works on principle of hybridization of mRNA (converted to cDNA) of concerned cell with immobilized cDNA/oligonucleotide sequence present on array slide

Microarray and DNA chips

• Advantage over SAGE: rapid evaluation of the comparison of two transcriptomes can be achieved by running them simultaneously on identical arrays and checking hybridization patterns of the two

• Further refined results can be achieved by using mRNA that is bound to ribosome in cell. Ribosome-bound mRNA gives part of transcriptome that is actively being used in protein synthesis

Difference between two

Microarray• Uses PCR products or

cDNA of the genes of interest

• Spotted on the surface of the glass slide or nylon membrane

DNA chip• The oligonucleotides

match positions within the gene of interest

• Mixture of oligonucleotides synthesized in situ on the surface of the glass slide or silicon membrane.

Objectives of Microarray and DNA chips

• Two major objectives

Objective: Identify

gene whose

mRNA is present

Microarray: PCR products or cDNA derived from all genes

of interestDNA chip: Mixture of

oligonucleotides synthesized which match

positions in the gene

Objectives of Microarray and DNA chips

Objective: Determine

relative amounts of mRNA present

Each position in array contains up to 109 copies of

probe molecules. Signal intensity at each position

will determine level of hybridization of probe with mRNA (high copy number,

more hybridization)

Basic Flowchart of Transcriptome study using Microarray/DNA chip

Collect mRNA molecules from

a cell

Use Reverse Transcriptase (RT)

enzyme to produce cDNA molecules from the mRNA

Label cDNA with flourescent dyes

Prepare microarray/DNA chip (cDNA from

reference genes or oligonucleotide

mixture)

Place labeled cDNA on

microarray slide

Hybridization of labeled cDNA

with cDNA (complimentary)

on microarray

Larger mRNA amount in cell

(more expression),

more hybridization.

Vice versa

Scan array slide. More

flourescence, more intensity of expression

Microarray and DNA chip method

Microarray/DNA chip after hybridization. Color intensity shows level of hybridization. The

cDNA prepared from mRNA is first labeled with fluorescent marker (like Cy3 and Cy5), then

hybridized with array to produce such a pattern.

Complications

• Hybridization analysis will have insufficient specificity to distinguish between every mRNA that could be present.Two mRNAs, similar

sequences, may cross-hybridize with

each other’s specific probe on

the array

Paralogous genes active in same

tissue – group of related mRNAs can

hybridize with members of the

same gene family

Distinguishing which specific

mRNA is present and how much is present becomes

difficult

Two or more different mRNAs could have been

derived from same gene – alternate splicing concept

Complications

• Alternate splicing

Complications

• When comparing more than one transcriptome, differences in mRNA amount and hybridization intensities must be due to difference in transcripts rather than due to experimental errors.

• Experimental errors could include:– Amount of target DNA on array– Efficiency with which probe has been labeled– Effectiveness of hybridization process

• Absolute precision and exact reproducibility of results is almost impossible in different laboratories doing same analysis (due to these experimental factors)

Normalization Procedures to Counter Experimental Factors

• To counter these experimental factors, certain normalization procedures are employed.

• Enables results from different array experiments to be accurately compared

• Normalization procedure:– Negative controls, so that background

can be determined in each experiment– Positive controls, always give identical

signals

Normalization Procedures to Counter Experimental Factors

• In vertebrates, actin gene is used as positive control– Its expression level is fairly constant in

any particular tissue– Even in developmental, or diseased

state

Alternative method to study and compare two transcriptomes

• Design experiment alternately• Compare two transcriptomes directly, on single

array • Label the cDNA with different flourescent probes• Scan the array at different wavelengths of light• Determine relative intensities of the two types

fluorescence at each position• Differences in the mRNA content of the two

transcriptomes can be directly analyzed.

Yeast transcriptome: An example

• Ideally suited for transcriptome studies• Little changes in yeast transcriptome, if

biochemical environment is constant• Glucose rich – stable. Glucose depletion:

causes corresponding restructuring of transcriptome.

• Transcriptome also undergoes restructuring during cellular differentiation.

• Sporulation pathway.

Yeast transcriptome: An example

• Spores adapt its mRNA at each stage to the changing stressful conditions.

• Acts as model organism to study interactions between genome and environmental signals in higher eukaryotes.

• Transcriptome studies help to annotate a genome sequence, helping in identifying gene functions.

Applications

• Studying the transcriptome can lead to various applications– Transcriptomes of stem cell and cancer cells

can be studied by researchers to understand cellular differentiation and carcinogenesis

– Transcriptomes of human oocytes and embryos can be studied to understand molecular mechanisms and signaling pathways in embryonic development

– Used in biomarker discovery

Thank you!