The overall goal of the following protocol is to enable genome scale interrogation of gene function by simultaneously monitoring multiple cellular readouts. In this demonstration, Firefly RAN and Cipro Dina Luciferase based reporters are used to monitor P 53 WINT and KRAS pathway activity respectively. These three reporters are cot transected with irna targeting a gene of interest into HCT one 16 cells, a colorectal cancer cell line.
Subsequently, firefly and vanilla luciferase activities are measured in the cell lysates while activity of Cipro DEA luciferase. A secreted enzyme is measured in the culture. Medium results are obtained that show the reproducibility of the assay system as well as the ability to reveal shared and unique actions of genes within these three cancer relevant pathways.
The main advantage of this technique over existing methods like microray or multicolor pries, that it is affordable, universal and cost effective way of looking at the transcription cellular outputs. This method can help to answer question in cancer research such as development of molecularly targeted therapeutic strategies. Although our transient transfect protocol effort IL and minimal upfront time commitment for screen setup, it'll likely increase well to well signal variability, which can be countered by looking at the report ratios instead of looking at absolute failures from the wells To begin this procedure, wash five 10 centimeter squared plates of 80 to 90%confluent HCT one 16 cells with PBS then harvest cells by trypsin ization for each plate.
Use one milliliter of trypsin solution followed by neutralization with 10 milliliters of DMEM containing 2%FBS and 1%penicillin. Streptomycin transfer suspended cells to a 50 milliliter conical tube and centrifuge at approximately 130 G for five minutes. Remove the supernatant and resuspend the cell pellet in 10 milliliters of media.
Count the cell number with a cell counter and then make a cell solution with 10 to the fifth cells per milliliter. Keep 150 milliliters of cell suspension for the next step using a multi drop automated liquid dispenser plate. 100 microliters or 10 to the fourth cells in each well of a 96 well culture plate.
The cell suspension in this case should be sufficient for plating 1296. Well plates incubate the plates with cells at 37 degrees Celsius in an incubator with 5%carbon dioxide while preparing the transfection mixture. The high quality reporter construct DNA used here was isolated using standard MIDI prep kits and diluted to a final concentration of one milligram per milliliter.
For each reporter, prepare 100 microliters of reporter construct stock solution by combining the following 30 microliters of P 53, FL 30 microliters of eight X-T-C-F-R-L 30 microliters of elk one G four six microliters of U-A-S-C-L and four microliters of H two oh. This will result in a DNA mixture with a final ratio of one to one to one to 0.2 of the reporters and a final DNA concentration of 0.3 milligrams per milliliter. Next, prepare I-R-N-A-D-N-A transfection mixtures sufficient for transecting 1296.
Well plates dilute 80 microliters of the reporter construct stock solution in eight milliliters of EC buffer from the QIAGEN effecting kit to obtain a DNA solution with a final concentration of three micrograms per milliliter plate. 20 microliters of the DNA solution into each well of a 96 well PCR plate. The number of 96 well plates will depend on how many irna pools are to be tested.
For example, in this case, we will need 4 96 well plates to evaluate. 384 different sir a pools using a biome automated liquid handler transfer two microliters of each five micromolar siRNA to the corresponding well of the PCR plate containing the diluted DNA stock. Next, add one microliter of enhancer solution to each well of the DNA siRNA plate.
Allow the enhancer reaction to occur at room temperature for five minutes. Then add three microliters of effecting transfection reagent to each well and incubate at room temperature for an additional 10 minutes. To stop transfection complex formation, add 10 microliters of DMEM containing 2%FBS and 1%penicillin streptomycin to each well of the PCR plate.
Retrieve the 1296 well plates containing HCT one 16 cells from the incubator. Transfer 10 microliters of the transfection mixture to each well of a 96 well plate containing the cells as each transfection will be performed in triplicate. The same mixture will be applied to two additional 96 well plates containing cells.
Incubate the cells with transfection mixtures at 37 degrees Celsius in a humidified environment with 5%carbon dioxide for 36 hours. 36 hours after transfection of cells. Luciferase activities are ready for measurement.
As this study incorporates multiple reporters, one secreted into the culture medium and two others expressed in the cell cytoplasm. Measurements will be obtained from both culture medium as well as a cellular lysate to detect Cipro Dina luciferase or CL activity in the culture. Medium replica plate.
20 microliters of a culture medium to a white opaque 96 well plate. Using the multi drop add 20 microliters of CL assay buffer to each well. Next, add 10 microliters of Cipro Dina Lucifer and substrate to each well and detect CL activity using a luminometer to detect firefly luciferase and vanilla luciferase activities, cell lysates must first be made.
Remove the culture medium from the cells by turning each plate upside down and tossing the medium in a sink. Remove remaining medium by gently tapping the upside down plate on paper towels. Add 30 microliters of one x passive lysis buffer to each well of cells.
Place the plates on a platform rocker set at medium speed for five minutes at room temperature. After five minutes, add 20 microliters of the luciferase assay reagent to each well of cells and immediately measure firefly luciferase activity using the luminometer. Next, add stop and glow reagent to quench the firefly luciferase activity and then measure luciferase activity.
Prior to multiplexing the robustness of the three different screening platforms was individually assessed. Indicated colorectal cancer cell lines were transfected with the eight XTCF PP 53 ta Luke or ALK one Firefly Luciferase encoding reporter constructs along with pools of siRNAs targeting WINT beta-Catenin P 53 or KAS respectively. Pathway relevant genotypes for each cell line are indicated.
The strength of each screening platform was assessed by the reproducibility of these induced effects on the relevant signal transduction pathway. As shown in these representative results, the betaine and siRNAs have no effect on the eight XTCF reporter activity in RKO cells. In contrast to DLD one and HCT one 16 cells, which are respectively deficient in the A PC pathway, suppressor protein and express an activated form of beca tenin.
Subsequently, it was shown that these reporters could be used together for simultaneous measurements of gene activity of the wind beta-catenin P 53 and KRAS signal transduction pathways using the indicated IRNA pools in these graphs. Relative luciferase activities are shown on the Y axis and siRNAs targeting the indicated gene of interest are shown on the X axis. For example, an siRNA pool targeting beta-catenin reduces wind beta-catenin pathway activity, while not significantly affecting the P 53 and KRA S pathways.
While attempting to develop a multiplex reporter system, it is important to optimize the cell line of interest for transfection region and the reporter signal intent.
