Techniques for Gene Expression Analysis Hüseyin Tombuloğlu

DD SSH Microarray Real-time PCR Deep Sequencing

Macroarray vs. microarray Allows only to detect 10 genes More then 10 genes,. The technique allows even genome-wide scale expression analysis Northern Blot RT PCR DD SSH Microarray Deep Sequencing

Microarray

“Determination of the CcpA dependent genes in A “Determination of the CcpA dependent genes in A. thaliana R54W using cDNA microarray analysis” Determination of the effects of a point mutation

Isolation of RNAs ( WT – Mutant ) Reverse Transcription Labeling with flour dyes HYBRIDIZASYON Cy3 (mutant) 560 nm Cy5 (wild type) 675 nm flour dyes used for labeling with cDNAs that hybridize their counterparts on the glass slide.

ArrayPro analysis scatter plot Computer analysis tecnique. Define the grids, subgrids and bad spot locations (fast analysis) Composite picture

MicroPrep MAIN FEATURES LOWESS normalization Merging of DNA microarray data from changing slide versions Outlier detection Slide qualility assessment Slide based steps PrePreP identify the bad spots ( ignored or bad cells) PreP normalization (only the control and target signals need to be identified) Experiment step PostPreP modified analyses

Lowess Normalization (per grid) box data Before Lowess (grid) Lowess fitted data shows normal distrubition

We visualized the quantified signals for each slide on scatterplots. And selected the best ones to continue our experiment. X-axis;Cy5 Y-axis;Cy3

Normalization of microarray data is necessary for minimizing systemic errors. After normalization the data from replicate slides was merged.

MicroPrep can output the merged slide data in tables that can be used by CyberT. CyberT uses the standart T-satistic to determine relavant regulated genes.

Visualization (genome2D)

R54W strain has inactive CcpA. about 163 genes are CcpA- dependent. 128 genes are subject to repression (up-regulated in mutant) 35 genes are subject to activation (down regulated in mutant)

Mikroarray advantage In a short time, genom-wide differences can be observed Disadvantage High cost Not applicable for any organism Chip-based technology

Genhunter Differential Display mRNA cDNA(s) Anchor Primer (3) 5’ UTR Coding region 3’ UTR AAAAAAAn UCG Reverse transcribe cDNA(s) 5’ UTR Coding region 3’ UTR AAAAAAAn UCG

cDNA(s) 5’ UTR Coding region 3’ UTR AAAAAAAn PCR Amplify labeled dNTPs UCG PCR Amplify labeled dNTPs

cDNA(s) Anchor Primer (3) 5’ UTR Coding region 3’ UTR AAAAAAAn UCG PCR Amplify labeled dNTPs

cDNA(s) Anchor Primer (3) 5’ UTR Coding region 3’ UTR AAAAAAAn UCG Arbitrary Primers (80) PCR Amplify labeled dNTPs

cDNA(s) X Y Z Anchor Primer (3) 5’ UTR Coding region 3’ UTR AAAAAAAn UCG Arbitrary Primers (80) PCR Amplify labeled dNTPs X Y Z

cDNA(s) X Y Z Anchor Primer (3) 5’ UTR Coding region 3’ UTR AAAAAAAn UCG Arbitrary Primers (80) PCR Amplify labeled dNTPs X Y Z Run on denaturing Polyacrylamide gel

cDNA(s) X Y Z Anchor Primer (3) 5’ UTR Coding region 3’ UTR AAAAAAAn UCG Arbitrary Primers (80) PCR Amplify labeled dNTPs X Y Z Run on denaturing Polyacrylamide gel

Constant Induced Induced

Constant Induced Induced

Gel extract PCR amplify

Gel extract PCR amplify Vektöre Ligasyon , transformasyon…miniprep ve dizileme

Sequencing and analyze Gel extract PCR amplify Vektor Ligation, transformation Sequencing and analyze

Differential Display-Farklılık Gösterim Tekniği Genhunter Advantages -Simple, gel based technology. - Easy to show the differentiated genes Disadvanteges - If there are a lot of proteins, it is hard to clone all the genes to the vectors and it is expensive.

Supressive Subtractive Hybridization (SSH)

Supressive Subtractive Hybridization (SSH) Reverse transkripsiyon RNA Sample RNA control Reverse transkripsiyon

Supressive Subtractive Hybridization (SSH) RNA Kontrol dokudan RNA Örnek dokudan Reverse transkripsiyon cDNA cDNA RE cut Adaptor ligation Denaturation and mix the RNAs

Supressive Subtractive Hybridization (SSH) RNA Örnek dokudan RNA Kontrol dokudan Reverse transkripsiyon cDNA cDNA Enzim kesimi Adaptör Ligasyonu Denaturasyon ve örneklerin karıştırılması cDNA pool cDNA pool

Supressive Subtractive Hybridization (SSH) RNA Örnek dokudan RNA from Control tissue. Reverse transkripsiyon cDNA cDNA Enzim kesimi Adaptör Ligasyonu Denaturasyon ve örneklerin karıştırılması cDNA subtracted out- remove

Supressive Subtractive Hybridization (SSH) RNA Örnek dokudan RNA from Control tissue. Reverse transkripsiyon cDNA cDNA Enzim kesimi Adaptör Ligasyonu Denaturasyon ve örneklerin karıştırılması PCR amplification, clonning, sequencing Amt cDNA subtracted out- çıkartılır

Supressive Subtractive Hybridization (SSH) Advantage -In a short time, it is possible to obtain hundreds of clones.

Supressive Subtractive Hybridization (SSH) Disadvantages - Junk sequences. - Expensive.

Real-time PCR Quantitative Real-Time Polymerase Chain Reaction(qRT-PCR) Multiplex Real-Time Polimerase Chain Reaction (MRT-PCR) RT-qPCR

Real-time PCR advantages * not influenced by non-specific amplification * amplification can be monitored real-time * no post-PCR processing of products (high throughput, low contamination risk) * ultra-rapid cycling (30 minutes to 2 hours) * requirement of 1000-fold less RNA than conventional assays (3 picogram = one genome equivalent) * detection is capable down to a 2-fold change * confirmation of specific amplification by melting curve analysis * most specific, sensitive and reproducible * not much more expensive than conventional PCR (except equipment cost)

Real-time PCR disadvantages * not ideal for multiplexing * setting up requires high technical skill and support * high equipment cost * DNA contamination (in mRNA analysis)

Real-Time PCR Principles Three general methods for the quantitative assays: 1. Hydrolysis probes (TaqMan, Beacons) 2. Hybridization probes (Light Cycler) 3. DNA-binding agents (SYBR Green)

Deep Sequencing

http://www.youtube.com/watch?v=77r5p8IBwJk

Deep Sequencing Organizmanın tüm transkriptomu çıkarılabilmektedir Günde 1 trilyon baza kadar sekanslama düşük maliyetle yapılabilmektedir. Bir haftalık süreçte 1.3 Gb büyüklükte data elde edilebilmekte. Genomik dizinin yüksek güvenilirlikte olması için aynı dizi defalarca analiz edilir. Konvansiyonel sistemlere göre (kapiller sekanslama ve elektroforez) hata oranı daha azdır.

Three Major Players 454 Roche SOLiD Applied Biosystems Genome Analyzer Illumina

The Roche 454/GS FLX Sequencing Technology 1. Örnekler öncelikle rastgele 300-800 b lık dizilere ayrılır

Pürifikasyon, amplifikasyon ve dizileme için gerekli adaptörler DNA parçalarının her iki yanına da eklenir. Eğer örnek çift zincirli ise bir zinciri uzaklaştırılır ve geriye kalan tek zincir ile devam edilir.

Adaptörler yardımı ile her bir ssDNA kendi özel boncuğu tarafından yakalanır. Boncuk ve bağlı fragment yağdaki su tanesi gibi davranış sergileyerek kendi içerisine kapanır ve bu sayede her bir fragment kendi içinde kontaminasyona uğramadan çoğalabilir. Bu etki emulsiyon etkisi: emPCR şeklinde adlandırılır.

4. emPCR her fragmenti milyon kez kopyalar. Amplifikasyon sonrası klonlar ortamdan arındırılır ve boncuklarla birlikte fiber-optic PicoTiterDevice cihazına dizileme için konulur..

5. The PicoTiterPlate her bir kuyuya bir boncuklu fragment alacak şekilde yerleştirilir.

Sequencing is accomplished by synthesizing the complementary strands of the bead attached templates. In a number of cycles the four bases (ATGC) are sequentially washed over the PicoTiterPlate. The incorporation of a new base is associated with the release of inorganic pyrophosphate starting a chemical cascade. This results in the generation of a light signal which is captured by a CCD camera. The released PPi is converted to ATP with adenosine 5′-phosphosulfate (APS) and ATP sulfurylase. The ATP is used by luciferase in the metabolism of luciferin, which emits photons.

Kaynaklar http://www.cals.arizona.edu/microarray/Jan07Workshop/lectures/Lecture%204.pdf Craig Tomlinson. DNA Deep Sequencing, PEMM Biostatistics, 2009 M. Tevfik DORAK, MD PhD, Real-Time PCR. 2010