Lecture 9 - Microarrays via Deep mRNA Sequencing PDF

MICROBIAL GENETICS Lecture 9 Global gene expression studies microarrays via deep mRNA sequencing Prepared by: Dr. Abeer Aloufi Assistant professor in Microbiology via Lecture 9 - Microarrays via deep mRNA sequencing ▪ Deep sequencing (also known as second-generation sequencing or next-generatio...

MICROBIAL GENETICS Lecture 9 Global gene expression studies microarrays via deep mRNA sequencing Prepared by: Dr. Abeer Aloufi Assistant professor in Microbiology via Lecture 9 - Microarrays via deep mRNA sequencing ▪ Deep sequencing (also known as second-generation sequencing or next-generation sequencing) differentiates it from the Sanger sequencing (also known as dideoxy or capillary electrophoresis sequencing) method ▪ In principle, the concepts behind Sanger vs. next-generation sequencing (NGS) technologies are similar ▪ In both NGS and Sanger sequencing, DNA polymerase adds fluorescent nucleotides one by one onto a growing DNA template strand ▪ Each incorporated nucleotide is identified by its fluorescent tag Lecture 9 - Microarrays via deep mRNA sequencing ▪ The critical difference between Sanger sequencing and NGS is sequencing volume ▪ While the Sanger method only sequences a single DNA fragment at a time, NGS is massively parallel, sequencing millions of fragments simultaneously per run ▪ This process translates into sequencing hundreds to thousands of genes at one time ▪ NGS also offers greater discovery power to detect novel or rare variants with deep sequencing Lecture 9 - Microarrays via deep mRNA sequencing Lecture 9 - Microarrays via deep mRNA sequencing ▪ Microarray can be used to determine gene expression patterns in a particular cell or tissue ▪ A DNA microarray allows scientists to experiment on thousands of genes at the same time ▪ Each spot on a microarray contains multiple identical strands of DNA ▪ The DNA sequence on each spot is unique ▪ Each spot represents one gene 10 100 nude W ▪ Thousands of spots are arrayed in orderly rows and columns on a solid surface (usually glass) ▪ The precise location and sequence of each spot are recorded in a computer database ▪ Microarrays can be the size of a microscope slide, or even smaller Oligo Lecture 9 - Microarrays via deep mRNA sequencing ▪ mRNA expression profiling using microarrays is a technology that allows simultaneous determination of the mRNA levels of many genes ▪ Microarrays range from small custom arrays designed to monitor expression of a few hundred genes to very large arrays that represent tens of thousands of genes or entire genomes ▪ Presently, there are three major applications for microarray data: Lecture 9 - Microarrays via deep mRNA sequencing ▪ One application treats microarray data as massively parallel expression assays. Typically, this data is used for the identification of particular genes that undergo expression changes in response to particular treatments, in particular cell types, or particular mutants ▪ A second application involves treating expression profiles as descriptions of collective behaviors. The state of the cell from which the sample was prepared is collectively characterized by determining the expression levels of tens of thousands of genes ▪ A third application that is becoming feasible is the mining of large expression profiling databases to characterize expression patterns of genes of interest over a wide range of tissue types, after various treatments, or in different mutants Lecture 9 - Microarrays via deep mRNA sequencing Strengths and weaknesses of expression profiling ▪ RNA and corresponding cDNA species have relatively homogenous chemical characteristics and can be specifically detected based on hybridization of complementary strands ▪ These advantages enable the simultaneous monitoring of tens of thousands of genes with very good sensitivity and accuracy at a reasonable cost ▪ Another advantage of using cDNA species is that they can be amplified by PCR and/or in vitro transcription ▪ Consequently, the amount of RNA required for expression profiling has been decreasing as methods for quantitative amplification of cDNA have improved Lecture 9 - Microarrays via deep mRNA sequencing Strengths and weaknesses of expression profiling ▪ On the other hand, the information obtained by expression profiling is simply the amount of each mRNA species present ▪ mRNA levels do not necessarily correlate with levels of active, properly localized proteins or the amounts of metabolites that they produce ▪ Consequently, the biological significance of observed changes in mRNA levels is open to question ▪ This limitation must be kept in mind when interpreting the results of expression profiling experiments Lecture 9 - Microarrays via deep mRNA sequencing Microarray Technologies ▪ In its first incarnation, microarray technology involved spotting random cDNA clones to detect genes with differential expression levels in different samples in organisms with very limited DNA sequence information ▪ The cDNA clones corresponding to genes that were found to be differentially expressed were then sequenced ▪ An early example is the differential display of mRNA, followed more recently by methods including serial analysis of gene expression (SAGE) and massively parallel signature sequencing (MPSS) Lecture 9 - Microarrays via deep mRNA sequencing Microarray Technologies ▪ Due to their capacities for covering large numbers of genes and rapid short sequence tag determination, SAGE and MPSS are also used for the discovery of new mRNA species in organisms where genome information is available ▪ Generally, the cost per sample of these methods is fairly high Lecture 9 - Microarrays via deep mRNA sequencing E. coli strain MG1655 E. coli BL21 Culture RNA isolation Reverse transcription and fluorescent tagging Hybridization onto microarray Not present in cells In E. coli strain MG1655 only Present in both cells In E. coli BL21 only Lecture 9 - Microarrays via deep mRNA sequencing Lecture 9 - Microarrays via deep mRNA sequencing Principle ▪ The principle of Microarray is based on: 1. Hybridization 2. The total strength of the signal from a spot depends upon the amount of target sample binding to the probe present on the spot Lecture 9 - Microarrays via deep mRNA sequencing Advantages and disadvantages Advantages 1. Microarrays allow the researcher to measure the relative quantities of specific mRNAs in two or more samples for hundreds or thousands of genes simultaneously 2. Fast and Easy to obtain the results Lecture 9 - Microarrays via deep mRNA sequencing Advantages and disadvantages Disadvantages 1. There is also a high cost associated with just a single experiment, as a single experiment can cost hundreds to thousands of dollars 2. DNA microarrays also lack sensitivity for genes expressed at low or very low levels and have a small dynamic range 3. The production of too many results at a time requires a long time for analysis, very complex