Time Series gene expression profiling of Lethal Giant Larvae tumours in developing wings of Drosophila larvae

Cancer is a complex phenomenon involving multiple cellular pathways and processes in various combinations. As a genetic system, Drosophila has contributed much to our understanding of processes that initiate tumorigenesis. However, these are still disparate threads of information and a comprehensive understanding of how these various events and processes come together to bring about tumorous transformation is still wanting. The present study is an attempt to create a comprehensive picture of the dynamic process of epithelial tumour development in imaginal discs using lgl mutants as a model of neoplastic transformation. Experimental Procedure: The experiments were carried out in isogenized w-; lgl4. FRT40A/CyO. Actin –GFP (E Gateff and Schneiderman 1974). Fairly homogenous cultures of flies were added to population cages 2-3 days post eclosion for egg collection. Eggs were collected on yeasted agar plates which were kept in the cages for 1 to 1.5 hours. Plates with eggs were kept at 25 degrees for 21 hours, after which they were gently washed under running water to remove early hatched larvae. The plates were then yeasted again and kept at 25 degrees for another 24 hours to let all the eggs hatch. The food with the 24 hour old larvae was then transferred to large food-grade plastic boxes that had a single cotton plugged hole on the lid to allow for proper ventilation. These boxes also had standard fly food and were well yeasted. The larvae were allowed to grow in these boxes at 25 degrees until the time they were harvested. No other synchronization process was done on these cultures. The boxes containing synchronized larval cultures were washed in gently running water and the water along with the washed off larvae were collected in a beaker. The larvae were allowed to sink to the bottom and the water was decanted off. This was repeated many times till the decanted water was clear of food and other debris. Finally, a 3M solution of NaCl was poured into the beaker. This causes the larvae to float to the surface along with the large pieces of food left over from the water washes. Water was then gently added to the beaker till the concentration of the salt solution was just enough to allow only the larvae to float on the surface while the food pieces sunk to the bottom. At this point, the solution, along with the floating larvae was decanted off through a sieve to collect the larvae. These were then transferred to a glass petri-plate and washed with distilled water to remove all traces of salt solution. The larvae were finally floated in PBS for GFP selection under fluorescence microscope. The selected larvae were transferred to glass cavity blocks rinsed in RNAzap RNAse Inhibitor solution containing RNA-ase free PBS and dissected. For isolating imaginal discs, larvae were cut from the middle with the help of forceps and scissors, and then flipped inside out with needles so that the inner body wall is completely exposed with the imaginal discs attached. Fat bodies and other internal organs were carefully removed with the help of the forceps and needles. The imaginal discs/tumors were dissected out from these flipped larvae, care being taken to remove all traces of fat bodies or other debris from the discs and immediately transferred to RNAse free Microfuge tubes. The tubes were centrifuged briefly to collect the imaginal discs/tumors at the bottom while the PBS was pipetted off. The discs were then resuspended in 150-300 uL of RLT Buffer (Qiagen) in which 1.5-3 uL of Beta-mercaptoethanol was added. The tubes were then vortexed to lyse the cells. This suspension was stored at -40 degrees until RNA preparation. Total RNA was prepared from the lysed wing discs/tumors suspended in the guanidine thiocynate containing Lysis Buffer (RLT) supplied with the RNeasy Mini Kit and 14.3M beta-Mercaptoethanol using the ethanol precipitation/column filtration protocol recommended for the RNeasy Mini Kit by the Manufacturer. The purified total RNA was quantified and its quality checked by spectrophotometry and Agarose Gel electrophoresis respectively. Total RNA from 30-50 pairs of imaginal discs/tumors were used to make a single sample depending on the tumor stage with RNA yields typically ranging from 10 ug to 15 ug. Purified total RNA passing the quality controls were immediately used for labeling and hybridization reactions. The total RNA was processed and hybridized to Affymetrix GeneChip Drosophila Whole Genome 2.0 arrays using the manufacturer’s protocol, and the arrays were scanned immediately after hybridization.