The overall goal of this procedure is to identify involvement of novel components of ubiquitin and ubiquitin like modifier systems in biological pathways, using the HF one alpha dependent hypoxia response as an exemplar. This is accomplished by first optimizing the response of the reporter cell line to the environmental stimulus hypoxia, so they're suitable for screening. The second step of the procedure is to establish the high and low controls to give a suitable Z factor for screening.
With the parameters established, the primary irna screen is performed in triplicate, after which hits are confirmed by secondary and tertiary deconvolution screens. Ultimately, the results can reveal new ubiquitin and ubiquitin like system regulators of the biological pathway in question. The main advantage of this technique over existing methods like whole Genome Irna screening, is that the targeting screen is faster, easier to perform, less expensive, and reports only on the members of the biochemical pathway in which the investigator is most interested To characterize.
The hypoxia responsiveness of the U2 O-S-H-R-E reporter cell line have ready 2 75 centimeters square flask of cells in DMM with 10%FBS. When the cells are at 80 to 90%Confluence aspirate the media from one flask and wash those cells twice with 10 milliliters of PBS. Tryps anize the cells and re suspend them in eight milliliters of DMM with 10%FBS make the cells homogenous with a pipette and obtain an average of two cell counts, then dilute the cells to 60, 000 per milliliter.
Next, using a multi-channel pipette, add 6, 000 cells to three columns of five white walled 96 Well assay plates incubate the five plates overnight while incubating to overcome the edge effects. Put a moist tissue paper at the base of the plates and place a stiff, transparent plastic bag over the stack of plates at the end of the following day. Note the time and transfer one plate to the hypoxia workstation set to 1%oxygen.
Then move a plate to the hypoxia workstation at 14, 18, and 22 hours after the incubation started. Next, make up 30 milliliters of luciferase lysis buffer. Be sure the BSA is allowed to dissolve for at least 30 minutes in the buffer before adding Lucifer and sodium pyrophosphate.
24 hours after the first plate edition, unload the hypoxia workstation and add 100 microliters of the prepared buffer to each loaded well of the assay plates to thoroughly lice the cells. Cover the plates with clear film and shake them for 10 minutes. At 500 RPM, now transfer the plates in a stack to the automated plate reader in the software.
Select the protocols menu and choose a BS 5 95. Then highlight all the wells to be read under the well selection menu. Click the run function and calculate the average reporter activity per plate.
Then normalize the data to the readings from the plate without hypoxia treatment. A robust response to hypoxia is essential for the assay and the tenfold increase in report activity is excellent. Proceed by setting the controls and calculating the Z factor.
The acid development stage is the most critical part of the screening procedure. It is important to have a robust phenotypic response of the cell line coupled with good high-end low controls. This will ensure the IRNA screen is a success.
Perform the primary screen in triplicate using the 96 well format. Seven confluent flasks of cells will suffice. Start by thawing out a dilution series of the SI RNA ubiquitin library for at least 30 minutes under a hood.
Next, make a master control plate with four 200 microliter replicates of 200 nano molar SI.RNAs. Load four wells with a high control four wells with a low control four wells with a buffer only control and four wells with a non-target IRNA control. Next label, 17 white walled assay plates between the control columns.
Each well gets loaded with a pool of four siRNAs targeting this same mRNA. To ensure the solutions in the master control plate and library plates are pooled to the bottom of each, well spin the plates for a minute at 2000 gs. Then using an automated dispenser transfer 10 microliters of each control well in the master plate to each of the labeled 17 plates.
Next, use the automated dispenser to transfer 10 microliters of each pool of SI RNAs from the library plates to an assay plate. Prepare 40 milliliters of transfection reagent at the predetermined ratio using the dispenser Transfer 10 microliters to each well of the 17 assay plates. 40 milliliters provides enough dead volume for the dispenser system.
After loading the transfection reagent, shake the plates for a minute at 500 RPM. Then allow them to incubate for 20 to 60 minutes to form the S-I-R-N-A transfection reagent complexes. Now wash 2 75 centimeter square flask of U2 O-S-H-R-E cells with 10 milliliters of PBS each.
Then tryps anize the cells and resuspend them in eight milliliters of DM EM with FBS, combine both cell collections in a 50 milliliter of plastic tube. Then calculate the cell density as before. Use the value to prepare 155 milliliter suspension of cells at 75, 000 per milliliter.
This volume is sufficient to load the plates and fill the dispenser's dead volume under the hood. Keep the cells stirred up with the magnetic stir. Using the automatic dispenser load 80 microliters of cells into each well of the reaction plates.
Each prepared reaction. Well now contains 100 microliters with the SI RNAs at 20 nano molar. Record the time and stack the plates in groups of five in a 37 degrees Celsius incubator.
Allow the plates to incubate for 24 hours over the next two days. Initiate the two replicates of these 17 reaction plates. 24 hours after the transfection transfer, the reaction plates to the hypoxia workstation.
Incubate the plates under 1%oxygen for 24 hours to induce the reporter the following day, two hours before the hypoxia period is complete. Prepare fresh luciferase lysis assay buffer as before 200 milliliters is enough for 17 plates. After removing the plates from the hypoxic environment, use the automated dispenser to load each well with 100 microliters of the assay buffer.
Then cover each plate with a clear film and load them into the shaker shaking at 500 RPM for 10 minutes. The cells will be thoroughly lysed. Now proceed with reading the plates as previously detailed.
Analyze the data taken in triplicate, and identify irna of interest for the subsequent screens. Greens U2 O-S-H-R-E cells contain a firefly luciferase reporter fused to three downstream copies of the hypoxia response element. These elements are bound by HIF one alpha HIF one beta heterodimers, which are abundant during hypoxia.
The hypoxia responsiveness of these cells was determined prior to screening with the I a library using the outlined protocol. 24 hours of hypoxia induced a tenfold increase in luciferase expression over baseline. Hi F1 alpha and FIH one I a were selected as the low and high controls cells.
Transfected with these controls showed 10%or 170%of the baseline luciferase signal after 24 hours of hypoxia. Next, the cells were transfected with ubiquitin IRNA library and exposed to 1%oxygen for 24 hours. In analyzing the reaction plates, a desirable Z factor of greater than 0.5 was obtained for each plate.
The values from each plate were displayed using a scatter plot, the primary screen registered values between the high and low controls. As expected, there were approximately equal numbers of positive and negative IRNA regulators, while most had no significant effect on the reporter, secondary and tertiary screens continued this study details on which are given in the text protocol. After watching this video, you should have a good understanding of how to perform a targeted irna screen to identify components of ubiquitin and ubiquitin lake systems that regulate your specific biological pathway of interest.
Thus providing a starting point for further investigations.
