Microarray Technique in Molecular Biology PDF

SSCG 2713 Genetic Engineering Technique in Mol. Bio: Microarray Dr. Nazrin Abd Aziz UTM KUALA LUMPUR Overview Microarray were introduced by Pat Brown and Ron Davis This technology use similar concept as Blotting A microarray is a laboratory tool used to detect the...

SSCG 2713 Genetic Engineering Technique in Mol. Bio: Microarray Dr. Nazrin Abd Aziz UTM KUALA LUMPUR Overview Microarray were introduced by Pat Brown and Ron Davis This technology use similar concept as Blotting A microarray is a laboratory tool used to detect the expression of thousands genes at the same time Principle of Microarray DNA microarrays are microscope slides that are printed with thousands of tiny spots in defined positions, with each spot containing a known DNA sequence or gene Often, these slides are referred to as gene chip or DNA chips The DNA molecules attached to each slide act as probes to detect gene expression Principle of Microarray CANCER CELL Genes Abnormal gene expression may result in cancer mRNA NORMAL CELL Microarray workflow Collection of samples mRNA isolation from samples of interest, followed by cDNA conversion cDNA labeling (Probing) Hybridization Data Collection and analysis 1. Collection of samples Two types of cells are collected Control Experiment Healthy cell Cancer cell Two types of cells are collected to compare the gene expression 2. mRNA isolation from samples of interest, followed by cDNA conversion RNA is extracted from the sample As mRNA has a poly-A tail, poly-T-tails are used to bind mRNA. mRNA then converted to cDNA Microarray workflow Collection of samples mRNA isolation from samples of interest, followed by cDNA conversion cDNA labeling (Probing) Hybridization Data Collection and analysis 3. cDNA labeling (Probing) A common labeling method used is fluorescent dyes, Cy3 & Cy5 The fluorescents offers several advantages such as high sensitivity, able to be detected simultaneously, and provide alternative for radioactive materials Different sample need to be labeled with different colors to distinguish specific group within same experiment 4. Hybridize the labeled target to the microarray The labeled cDNA from both samples are placed in the DNA microarray so that each cDNA gets hybridized to its complementary strand, they are also thoroughly washed to remove unbounded sequences. After washing off non-specific bonding sequences, only strongly paired strands will remain hybridized. 5. Data Collection and analysis The collection of data is done by using a microarray scanner. When the laser scans the array, the camera records the images produced. Then the computer stores the data and provides the results. The produced data are then analyzed. The difference in the intensity of the colors for each spot determines the character of the gene in particular spot. Microarray workflow Collection of samples mRNA isolation from samples of interest, followed by cDNA conversion cDNA labeling (Probing) Hybridization Data Collection and analysis Video https://www.youtube.com/watch?v=0ATUjAxNf6U