mRNA Analysis

Expression Microarrays

Even though each cell in the human body contains identical (or nearly identical) DNA, every gene is not expressed in the same way in every cell. Study of the differential expression of genes helps to elucidate how cells are affected by developmental or environmental changes. To understand the complex mechanisms and networks involved in biological processes and diseases, it is no longer sufficient to focus on isolated pathways or single genetic events. Instead, scientists are embracing a systems biology approach for a more complete knowledge of intricate regulatory networks and complicated pathways. With the development of expression microarray technology, it is possible to examine the activity of thousands of genes at any given time.

Thousands of gene sequences are printed onto a single microscope slide, resulting in a microarray. To determine which genes are turned on or off in a given cell, mRNA is extracted from a control and a variable cell and differentially labeled with fluorescent dyes. The control and variable sources of mRNA could be normal vs. tumor cells, or cells experiencing or not experiencing vibration. These labeled products are allowed to competitively hybridize to the microarray. If a particular gene is highly expressed, it produces many molecules of mRNA, which hybridize to the microarray generating a very bright area of a given fluorescent dye. If a normal transcript were labeled “green” and the same gene were labeled “red” in the variable source (i.e. tumor), and gene expression were lost in the variable source, then the spot would appear more green. Equal amounts of each give a “yellow” signal. We are utilizing microarray technology to develop expression profiles of laryngeal cells experiencing various stimuli at several stages of development.

Microarray Overview

Quantitative Real Time RT-PCR (Q-RT-PCR)

Quantitative detection of gene expression is possible using the fluorogenic 5' exonuclease assay. mRNA transcripts are reverse transcribed into cDNA which is used as the template in a modified PCR. A fluorogenic probe complementary to the target sequence is added to the PCR reaction mixture. The probe consists of an oligonucleotide with a reporter and quencher dye attached. During PCR, if the target of interest is present, the probe anneals specifically between the forward and reverse primer sites. The nuclease activity of the polymerase cleaves the probe, which releases the reporter from the quencher dye giving an increase in the fluorescence intensity of the reporter dye. This process occurs in every cycle and does not interfere with the accumulation of PCR product. To induce fluorescence during PCR, laser light is distributed to the 96 sample wells via a multiplexed array of optical fibers. The resulting fluorescent emission returns via the fibers and is directed to a spectrograph with a charge-coupled device (CCD) camera. We are using Q-RT-PCR to study the changes in expression of several genes differentially expressed in laryngeal cells experiencing vibrational stress.

Q-RT-PCR Overview