Parallel human genome analysis: Microarray-based expression monitor of 1000 genes 班級：生科四乙 指導老師：藍清隆 姓名：郭瑜芬 Mark Schena, Dari Shalon, Renu Heller, Andrew Chai, Patrick O. Brown, and ronald W. Davis

Introduction

1.The microarrays they used containing 1046 human cDNAs of unknown sequence which came from T cell were printed on glass. 2.The cDNA “chips” were uesed to quantitatively monitor differential expression of human gene which were under different condition. 3.Finally, they characterized those genes by sequencing.

In this article, the authors wanted to tell us: 1.The identification of known and novel heat shock and phorbol ester-regulated genes in human T cell demonstrates the sensitivity of the assay. 2.Parallel gene analysis with microarays provides a rapid and efficient method for largescale human gene discovery

What is “microarray”?

What is “T cell”?

Method and result

Part a. How many hybridization signals can observe in these cDNA array?

Method DNA source Prepare Probe cDNA Prepare Target cDNA LabelingMicrospotting Hybridization Microarray Detection and analysis Jurkat cell’s cDNA

To make the cDNA libary Reverse transcription cDNA ΛYES-R cDNA

To infect into E. coli and propagate JM107/ λ KC Propagate Cell lysisPCR amplification

Microarray preparation ﹡ A total of 1056 cDNAs, representing 1046 human clones and 10 Arabudiosis controls.

To prepare probe and label To isolate Jurkat mRNA Reverse transcrioption +Cy5-dCTP Probe ﹡ Arabidopsis control mRNAs were doped into the reverse transcription reaction at ratios of 1:100000,1:10000,1:1000(wt/wt) Jurkat cell 25 ℃ 43 ℃

Result

Conclusion 1.Hubridzation signals were observed to >95% of the human cDNA array element, but not to any of the Arabidopsis. 2.Fluorescence intensities spanned more than three orders of magnitude for the 1046 array elements surveyed.

Part b-1. In heat shock, how many genes can be active or repress and what are they?

The pathway of heat shock in T cell

DNA source Prepare Probe cDNA Prepare Target cDNA LabelingMicrospotting Hybridization Microarray Detection and analysis Jurkat cell’s cDNA Method

To prepare probe and label Jurkat cell 25 ℃ 43 ℃ To isolate Jurkat mRNA +Fluorescein +Cy5-dCTP Probe Reverse transcription

Result(I)

Result(II)

Part b-2. The result of the microarray at part b-1 is correct?

RNA source Prepare Probe RNA Prepare Target RNA LabelingSpotting Hybridization Detection and analysis Method

RNA dot plot Hybridiztion +probe

Result

Part c-1. In phorbol ester treatment, how many genes can be active or repress and what are they?

The pathway of PKC activation in T cell

Phorbol ester In the experiment, the purpose of phorbol ester is active protein kinase C.

DNA source Prepare Probe cDNA Prepare Target cDNA LabelingMicrospotting Hybridization Microarray Detection and analysis Method

To prepare probe and label Jurkat cell Untreat +Phorbol ester(4hr) To isolate Jurkat mRNA +Fluorescein +Cy5-dCTP Probe

Result(I)

Result(II)

Part c-2. The result of the microarray at part C-2 is correct?

Method RNA source Prepare Probe RNA Prepare Target RNA LabelingSpotting Hybridization Detection and analysis

Result

Part d. Can microarray be used to monitor expression in human tissue?

Method RNA source Prepare Probe mRNA Prepare Target RNA LabelingSpotting Hybridization Detection and analysis

To prepare probe and label 1.Human tissue Bone marrow Brain Prostate heart transcription +Cy5-dCTP Probe 2.Jurkat cell transcription +Fluorescein Probe

Method