An In Vitro Protocol for Evaluating MicroRNA Levels, Functions, and Associated Target Genes in Tumor Cells

To access neural microRNA target interactions with MicroRNA functions is critical to understand how MicroRNAs regulate various biological processes in both healthy and diseased states. This protocol describes the reproduce through strategy to identify microRNA targets and the functional microRNAs as the validation of direct target of microRNAs is challenging. Our protocol can provide insights on the function of individual microRNAs and the implication of a microRNA in cancer therapy.

Calculation of the half-maximal inhibitory concentration is an important method, not only for microRNA studies but for the efficacious evaluation of other anti-cancer drugs. Our protocol will be helpful to new researchers since we described the fundamental method to understand the level of microRNAs in a cell. To begin the protocol, count the cells with the cell counter.

Plate the cells in a 96 well plate. The next day, prepare a set of transfection mixtures to transfect the cells in several final concentrations of microRNA control mimic, and microRNA-107 mimic. From a stock of 25 micromolar microRNA control mimic, or microRNA-107 mimic, dilute and add control mimic or microRNA-107 mimic in the reduced serum media along with the transfection reagent in microcentrifuge tubes.

Gently mix the oligo-containing mixtures using a micropipette. After a 10 minute incubation in the cell culture hood, gently mix the oligo-containing mixtures again, and then add 50 microliters of the mixtures into each well. Keep the transfected cells in a cell culture incubator.

Carefully aspirate the cell culture media in each well of the plate and promptly fill 100 microliters of 10%trichloroacetic acid, or TCA, into each well. Keep the plate containing 10%TCA at four degrees Celsius for one hour. Next, wash the plate several times by submerging it into a tub with tap water.

Remove excess water from inside of the wells by tapping the plate until there is no water left, and dry the plate. Pipette 50 microliters of 0.4%SRB solution into each well including the blank wells. Gently shake the plate until the 0.4%SRB solution evenly covers the bottom of the wells.

Then, incubate the plate for 40 to 60 minutes. After incubation, wash the plate with 1%acetic acid until the unbound dye is totally washed away. Pipette 100 microliters of 10 millimolar Tris base solution into the corresponding wells, including blank wells.

Keep the plate on a shaker for 10 minutes. Measure the absorbance at 492 nanometers. Calculate the percentage of average absorbance and the standard deviation of each group using absorbance values of the SRB assay.

Import the raw data including treatment concentrations, average absorbance, and standard deviation into the software by vertically aligning the data. Click the Create Graph tab and choose the Simple Scatter Error Bars. Select worksheet columns as Symbol values, and click next.

In the Data Format panel, select X-Y pairs and click next. Select corresponding data columns in the Select Data panel. Click finish to create the plot.

Double-click on the X-axis to modify the type of scale in the scaling of axis. Change the type of scale from linear to log. Modify the start and end range number to 0.01 and 200, respectively.

Right-click on any scatter plot, choose Curve Fit, and go to the subcategory User Defined. Select dose response curve. Click the next buttons, and then click finish.

Go to the report tab, and then check the n, k, and R values. Run the PCR as detailed in the accompanying text protocol. Then move on to double-digestion by combining the reaction mixtures, including the restriction enzymes Exo1 and Not1 one in a tube.

Incubate the mixtures for three to four hours in a 37 degree Celsius water bath. Run the double-digested products on a 1%agarose gel. Then cut the bands under UV light.

Purify the double-digested PCR products and luciferase vectors from the excised bands. Continue with ligation of the PCR products into the luciferase vectors by preparing 20 microliters of ligation reaction mixtures including the DNA ligase. Briefly centrifuge the tube for 10 to 15 seconds.

Incubate the ligation at 16 degrees Celsius overnight using a thermal cycler. The next day, perform transformation by adding the ligation mixtures into the tube containing competent cells. Gently tap the tube and keep it on ice for 20 minutes.

Quickly and gently transfer the tube to a heat block at 42 degrees Celsius for 30 seconds to one minute. Following heat shock, place the tube on ice for 20 minutes. Spread competent cells on an LB agar plate.

Grow competent cells in an incubator at 37 degrees Celsius overnight. The following day, pick an individual colony and resuspend E.coli in one of the 8-strip tubes containing ultrapure water, and repeat this step to resuspend E.coli from four to eight randomly-selected colonies. Transfer 25 microliters of E.coli suspension into another set of 8-strip tubes.

Perform the colony PCR using the E.coli suspension. Prepare the luciferase assay in a 24 well plate. Add one to two times ten to the four cells in a 500 microliter cell culture media for each well.

After overnight incubation, transfect 50 nanograms of luciferase vectors into the cells with control mimic or a specific microRNA mimic using a transfection reagent. The next day, wash the inside of the wells twice using phosphate buffered saline. Apply 200 microliters of lysis reagent into the wells and sufficiently carry out cell lysis before measuring the luciferase activity.

Keep the plate shaking for at least 15 minutes. Transfer five to 10 microliters of cell lysate into the new tube, and add 100 microliters of reagent one and immediately mix the solution by pipetting. Then read the firefly luciferase activity using illuminometer for 10 to 15 seconds.

Add 100 microliters of reagent two in the same tube and then mix by pipetting twice. Finally, read the renilla luciferase activity for 10 to 15 seconds using illuminometer. RTPCR shows a significant reduction of microRNA-107 and PANC-1 and CAPAN-1 cells compared with HPNE cells.

The levels of microRNA-301 were significantly upregulated in PANC-1 and CAPAN-1 cells, compared with HPNE cells. The SRB assay in this study clearly demonstrates that the proliferation of PANC-1 cells decreased following a microRNA-107 mimic transfection. The screening of 11 potential predicted targets of microRNA-107 clearly shows that only synuclein gamma, or SNCG, a positive regulator of tumor cell growth directly interacts with microRNA-107 and PANC-1 cells, indicating that microRNA-107 can negatively regulate the proliferation of PANC-1 cells by modulating the SNCG expression.

Adult cell proliferation assays that use the tetra hydroleum-based entity and CFSE are applicable to screen the effect of such as microRNA mimics, depending on the cell types. Based on the experimentally-validated function of microRNAs, a systematic review of database and target prediction program is required to narrow down the list of candidate targets before luciferase assays. For the evaluation of combination efficiency of microRNAs with anti-cancer drugs, it is advantageous to calculate adjusted IC values based on our protocol for the assessment of combination index.