Local background

Local background

Array level quality control

•Problems:

–array fabrication defect

–problem with RNA extraction

–failed labeling reaction

–poor hybridization conditions

–faulty scanner

–

•Quality measures:

–Percentage of spots with no signal (~30% excluded spots)

–Range of intensities

–(Av. Foreground)/(Av. Background) > 3 in both channels

–Distribution of spot signal area

–Amount of adjustment needed: signals have to substantially changed to make slides comparable.

Introduction Microarray Analysis

Arabidopsis thaliana is a small flowering plant that is widely used as a model organism in plant biology. Arabidopsis is a member of the mustard (Brassicaceae) family, which includes cultivated species such as cabbage and radish. The benefits of using this plant in biology are the small genome size (125 Mb), the extensive chromosome maps, short life cycle, and the large number of mutant line resources available (NIH 2012). All of these properties make it an ideal candidate for study using microarrays. The specific proteins of interest in our study, Beta-amylase or BAM proteins, work to cleave the amylase bond in starch molecules to catalyze its breakdown to maltose, a sugar dimer made of two glucose molecules. Specifically, it catalyzes the hydrolysis of the second alpha-1,4 glycosidic bond, cleaving off two glucose units at a time. Starch is accumulated during the day while the plant is exposed to the sun and then broken down at night when photosynthesis cannot occur. Our particular interest lies in a protein of unknown function, BAM-9. Previous studies have shown that BAM9 is located in the chloroplast, differing from the other locations of the BAM enzymes, and that its expression peaks during the nighttime hours. Other studies have found that the active site of BAM9 is different from the other BAM enzymes as well as that it does not appear to have any starch degradation activity, leading to the inference that BAM9 is not likely to be directly involved in starch degradation. This may mean that BAM9 has a surface binding site and may serve as a signaling protein during the end of starch degradation (Monroe, 2011).

                                                                                

                                                         

A microarray is a molecular biology technique that is used for studying cell-wide transcriptional changes. Scientists can use microarrays to measure the expression levels of a large number of genes simultaneously. A DNA microarray is a glass slide containing many different short pieces of DNA complimentary to each gene in an organism in a known position. Typically, RNA of interest is isolated and reverse transcribed to cDNA of interest and then hybridized to the microarray. The wild-type cDNA is labeled with a colored marker (red) while the knockout or mutant cDNA is labeled with a marker of a different color (green). The array is scanned first for red and then for green. The two are compared to see what genes are expressed in each plant and what the differences and similarities are (Rife 2012).

                                                                                                           

    In this experiment, a microarray was used to measure expression levels of a BAM-9 knockout versus a wild type genome. This information would tell us more about the proteins involved and the expression level changes, as well as the role they play in the organism. Specifically, we looked at genes expressing proteins in the chloroplast, which is where BAM-9 is thought to be located. This would help us to understand how a knockout of BAM-9 would alter specific gene expression levels and RNA levels, which would subsequently influence protein translation.