Microarray - created from Mind Map

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genomics and genome sequencing<br><br><ul><li>• The enAre human genome has now been sequenced

∼this involves 3 billion (US) base pairs of DNA

• This encodes 20,000 – 25,000 protein-coding gene sequences

~60% of human genes have spliced variants (2.7 on average)

∼there are about 100,000 different polypepAdes encoded in

human genome

• The funcAons of &amp;gt;75% of these gene sequences are currently

unknown

Genomes and Genome Sequencing

High throughput roboAcs have revoluAonised how we study

the role and interacAons of these genes efficiently</li></ul>

applications of technology and genome discovery<br><br><ul><li>Genome Discovery

High throughput genome sequencing of uncharted species of any kind

to study microbial biodiversity, ecology, and evoluAon. </li></ul>

transcriptomics<br><br><ul><li>The study of the total mRNA / ncRNA content of an

organism &amp;amp; or its consAtuent cells and Assues

simultaneously </li><li>This technology has improved/accelerated research in

medical, biotechnology, biosecurity, horAculture,

agriculture, aquaculture etc over the last 2 decades.</li></ul>

principle of microarray-labelled cDNA<br><br><ul><li>making cDNA flueorescent we design a prode, add flourescently labelled cDNA, hybirdise, laser excite and then detect </li></ul>

printing a typical array

Cahllenge

what can be compared?<br><br><ul><li>• Cancer research: Tumour / normal

• Complex diseases: Disease / non-diseased

• Time sequence

• Cell division

• Development

• Different Assues/bio-specimen

• Biodiversity / biosecurity

• Microbial species/ environmental samples

• Agricultural genomes

• Vine varietals

• Yeast species in beer </li></ul>

avaliable technologies

Case Study 1<br><br><ul><li>1. Cardiac hypertrophy can lead to heart failure.

2. Growth of heart can be:

i) physiological eg postnatal, chronic exercise

ii) pathological eg pressure/volume overload, myocardial

infarcAon (MI).

3. Pathological condiAon is associated with fibrosis and cardiac

dysfuncAon (increased morbidity and mortality) </li></ul>

technology

human genome

microarrays in studying canditate genes in disease/biological problems<br><br><ul><li>Candidate genes (genome scans, SNP chips)

∼possible disease-causing genes are idenAfied.

∼pedigree collected with linkage analysis </li><li>• PopulaAon studies (case-control, genome scans, SNP chips )

∼predisposiAon and severity of a disease, confounding factors

eg lifestyle, environmental factors. </li><li>• FuncAonal models (toxicology &amp;amp; physiology, usually animals)

∼observaAon of phenotype vs treatment extrapolated to human

diseases. </li><li>• Gene expression (in situ hybridisaAon, microarray &amp;amp; qRT-PCR)

∼quanAficaAon of mRNA, ncRNA copies </li><li>• Post-Genomics era:

∼Proteomics, pharmacogenomics, metablonomics, personalised

medicine, everything ‘omics’ </li></ul>

candidate genes

population studies

functional models

gene expression

microarrays

gene for which association with syndrome X reported

reversibility

DNA hybridisation<br><br><ul><li>DNA hybridization generally refers to a molecular biology technique that measures the degree of genetic similarity between pools of DNA sequences. It is usually used to determine the genetic distance between two organisms.</li></ul>

treatment<br><br><ul><li>labelled red to represent treatment</li></ul>

control<br><br><ul><li>labelled green </li></ul>

quantification according to fluorescence<br><br><ul><li>if more treatment quantified, red output, if more control wuantifies, green output, uf equal, yellow spot </li><li>each dot has DNA from a different gene and is ~11 microns </li></ul>

quantifying copies<br><br><ul><li>nrighter flourescence = high amount of mRNA no fluro = no mRNA </li></ul>

1st gen spotted array

what is printed on an array<br><br><ul><li>• SyntheAc short DNAs “oligonucleoAdes”

– (Greek “oligo, a few)

• PCR copies of DNAs

• Customised/designer oligonucleoAdes

• Anything that can hybridise…

• Other types of microarrays: protein, Assue etc</li></ul>

oli

oligonucleotides

PCR copies of DNAs

designer oligonucleotides

anything that can hybirdise

other types of microarrays

spot genes with changed expression

computational challenges<br><br><ul><li>Raw data files:

1. Spoxed array text file is ~30,000 kb excl image file ~30,000kb

2. Affymetrix array dx file is ~1Gb incl chip file

3. Agilent array text file is ~750Mb

• ComputaAonal power to compress data for analyses.

• StaAsAcs: mulAple comparison and modelling will require more

substanAal power.

* New sequencing technology – to sequence the whole genome

- Illumina Hiseq, Hiseq X Ten, MiSeq, NextSeq

- PacBio RS, NanoPore, Ion Torrent

- Roche 454 deep sequencing (whole genome sequencing)

eg Sequencing 20 million bases per 5 hour run

Terabytes of data (1000Gb) </li></ul>

moores law<br><br><ul><li>number of transistors on a chip will double every 18 months</li></ul>

SNPs

cHG

promoter

exon/splice variants

microRNA/IncRNAs

strategies for transcriptome based experiment

cardiac hypertrophy

shortening phenotype and gene function

validation of miR-34a expression

PI3K p110α<br><br><ul><li>PI3K p110α allows the heart to enlarge in response to

physiological sAmuli while maintaining normal or

enhanced funcAon</li><li>PI3K regulates cardiac

fibrosis - dnPI3K showed

increased fibrosis. </li><li>Gene expression of a

number of extracellular

matrix- and fibrosisrelated

genes were

upregulated in dnPI3Ks. </li></ul>

6 mouse model<br><br><ul><li>Transgenic mice with consAtuAvely acAve

(ca) PI3K mutant have 6.5 fold increase in

PI3K acAvity and 20% heart weight/body

weight raAo

§ Tg with dominant negaAve (dn) PI3K

mutant: 77% decreased PI3K acAvity and

HW/BW raAo is reduced by ~20%

§ Myocardial infarcAon (MI) performed at 4

months of age – occlusion of lec anterior

descending coronary artery.

§ Sham operaAon involved open chest

surgery. </li></ul>

dosage scheme for LNA-antimiR

presence of caP13K<br><br><ul><li>caPI3K-consAtuAvely acAve ↑ PI3K acAvity

dnPI3K-dominant negaAve ↓ PI3K acAvity </li><li>Presence of caPI3K rescues cardiac funcAon in the myocardial infarcAon (MI) seÄng. </li></ul>

regulating microRNA

hierarchal clustering showing distinct expression<br><br><ul><li>Gene signature reflected disAnct

cardiac stress in the 6 mouse

models assayed.

Significant &amp;amp; InteresAng Enriched

GO terms:

§ Mitochondrial inner membrane

§ Oxidoreductase acAvity

§ Lipid metabolic processes

§ Electron transport

§ Collagen </li></ul>

delivery

technical considerations

advantages

disadvantages