The overall goal of this procedure is to co transfect reporter constructs with a micro RNA mimic to validate predicted micro RNA targets in human three prime Ts.This is accomplished by first laying out an experimental design including choosing a cell line and selecting micro RNA mimics along with experimental and control three prime UTR Go Clone reporter constructs The adherence cells are then seated approximately 24 hours prior to transfection. The next step of the procedure is to co transfect each experimental or control three prime UTR go clone reporter construct with a micro RNA mimic or non-target and control, followed by a 24 to 48 hour incubation. As a final step, the light switch luciferase assay reagent is added directly to the cells and culture and the Lumin scent signal is read on a plate luminometer.
Ultimately results can be obtained that show which human three prime urs are targets of a given micro RNA through measurement of luciferase signal knockdown with reporter assay technology. This method can help answer key questions for micro RNA researchers because it provides a functional assay to validate predicted micro RNA three prime UTR target interactions in living cells. Experimental design begins with the selection of an appropriate highly transfect adherence cell line micro RNA mimic based assays work best in cell lines with low endogenous expression levels of the micro RNA.
Then select the micro RNA mimic and non-target control pair, including a non-target control is critical as cot transecting a plasmid and a small RNA oligo leads to lower signal than transecting the plasmid alone. Next, select the pre clone three prime UTR Go Clone reporter constructs from switchgears genome wide collection. Go to the online catalog and click on the UTR search button.
After entering the list of gene names, gene symbols, or accession numbers, the search result will provide vector and sequence information along with inventory status. Now select the control reporter constructs. Choose control constructs with three prime ur, unlikely to be targeted by the chosen micro RNA such as housekeeping, urs, or random fragments to control for non-specific effects of micro RNA overexpression on overall cell health or assay signal.
Also consider using a synthetic micro RNA target reporter as a positive control for mimic activity on day one seed low passage adherence cells, so they'll be 80 to 100%confluent for transfection 24 hours later. Using highly con fluent cells is key to achieving good results. Seed the cells in a white 96 well tissue culture plate in 100 microliters of total volume.
In parallel. See the appropriate number of cells in a clear plate for a sudden confluence On day 2 24 hours post cell seating. Prepare for transfection thaw.
Go clone reporter constructs and micro RNAs at room temperature. Once thawed mixed well dilute the micro RNAs to a working concentration of two micromolar in RNAs free water goone plasma DNA is delivered at 30 nanograms per microliter and does not require further dilution. To prepare the transfection mixture, consider that each D-N-A-R-N-A combination should be transfected in triplicate volume should be scaled to account for the number of assays being performed.
Create the transfection reagent mixture by combining 9.85 microliters of serum free media and 0.15 microliters of Dharma effect DUO per transfection. Allow the mixture to incubate at room temperature for five minutes. Then create the D-N-A-R-N-A mixture for each assay by combining 3.33 microliters of the go clone reporter construct with enough micro RNA mimic to achieve the desired final concentration.
Bring the volume to 10 microliters with serum free media. Next, combine 10 microliters of the transfection reagent mixture with 10 microliters of each D-N-A-R-N-A mixture and mix well. There is a final volume of 20 microliters for each assay.
Well cover and incubate for 20 minutes at room temperature. After incubating, add 80 microliters of prewarm antibiotic-free. Complete media for a total volume of 100 microliters per individual well are assay resulting in final mimic concentrations between 10 and 15 nanomolar.
As a final step, carefully remove the media from each well of seeded cells and replace with 100 microliters of the appropriate final transfection mixture. Return the plate to the incubator and incubate for 24 to 48 hours. Most of the mix will produce significant results within 24 hours.
A 48 hour incubation period will result in higher well to well variation in signal On day 3 24 hours post transfection conduct luciferase assays using light switch luciferase assay reagents cells may be assay immediately or frozen at minus 80 degrees Celsius and assay later. Adding the freeze thaw cycle will result in enhanced cell lysis and higher reporter signal. Bring the plate of transfected cells to room temperature.
Then prepare the light switch luciferase assay solution according to the kits instructions and pour into a sterile reservoir. Use a multi-channel pipetter to add 100 microliters of light switch assay solution directly to each sample. Well in the white 96 well plate cover the plate protect from light and incubate for 30 minutes at room temperature following incubation.
Read each well for two seconds on a plate Luminometer the knockdown of three prime UTR Luciferase reporter activity by LE seven A was measured in HT 10 80 cells by cot. Transecting each reporter construct with a let seven A mimic or non-target control to analyze the results. Consider the signal from the non-target control to be 100%for each construct.
Calculate the percent knockdown by dividing the signal from the specific micro RNA mimic by the non-target control signal. Any given micro RNA mimic may have non-specific effects on overall cell viability or assay signal to correct for any non-specific effects. Compare the knockdown observed with an experimental UTR target to the knockdown observed with housekeeping or random control T as shown here.
The PONC and arid three B human three prime urs are targets of the let seven A micro RNA signals from human three prime UTR reporters for the PUNC and arid three B genes are significantly knocked down in the presence of the let seven A micro RNA mimic. While signals for housekeeping and random sequence URS are not. After watching this video, you should have a good understanding of how to set up a successful cot, transfection of microRNA mimics and three prime UTR reporter constructs in living cells and read the signal on luminometer to measure signal lockdown.
