Detection of miRNA Targets in High-throughput Using the 3'LIFE Assay

The overall goal of the following experiment is to identify micro RNA targets amongst a library of three prime untranslated regions or UT R.This is achieved by COT transecting, HEK 2 93 cells with a micro RNA expression plasmid and a plasmid containing a luciferase reporter fused with a three prime UTR to facilitate large unbiased screens. Transfect are performed in 96 well plates as a second step. The cells are subjected to a dual Lucifer assay, which measures post transcriptional regulation of the three prime UTR by the micro RNA.

Next data from multiple replicates and multiple plates are analyzed In order to identify high confidence micro RNA targets amongst a pool of hundreds of three prime URS results are obtained that show each micro RNA targets a unique set of three prime UR based on translational repression as measured by the dual Lucci assay. The three-prime life essay can answer key questions in Micron, a biology by identifying a large number of target genes for a micron A of interest yielding data to direct downstream validation experiments 24 to 48 hours prior to transfection see a sufficient quantity of HEK 2 93 T cells based on the number of 96 well plates to be transfected. Each 96 well plate requires 9 million cells.

In this demonstration, two well-characterized microRNAs will be screened against the panel of 275 human three-prime urs. Hence 3 96 well plates will be used. A 145 millimeter circular culture plate is typically sufficient for 3 96.

Well transfect when cells are grown to approximately 90%co fluency, all buffers and plasmid DNA should be prepared prior to transfection. The human three prime UTR clones are resuspended to approximately 100 nanogram per microliter per well of each 96 well plate and the life micro RNA vectors are resuspended to a concentration of 500 nanograms per microliter. For each micro RNA and blank control plasmids reagents for making the 10 x firefly lucifers buffer and the one x vanilla lucifers buffer should be prepared before on the day of transfection, prepare a transfection buffer containing PBS and 1.5%HEPs at pH seven in formulating buffer and plasma DNA volumes.

Assume 120 reactions for each 96 well plate to sufficiently account for errors in pipetting and volume loss from using liquid reservoirs and multichannel pipettes. Prepare three stocks of P life micro RNA plasmid plus transfection buffer. For each micro RNA, this stock should account for 50%or 10 microliters of the total volume of each well multiplied by 120 wells.

Add 200 microliters of DM EM supplemented with 10%FBS and 1%pent strapp to each well of 3 96. Well cell culture plates. Place the plates in a 37 degree Celsius incubator for use following transfection.

Remove the HEK 2 9 3 T cells from the 145 millimeter culture plate by eluding the media washing gently with PBS and treating with approximately five milliliters of point 25%trypsin for five minutes at 37 degrees Celsius. After counting the cells aliquot to three tubes each containing 9 million cells, spin cells at 300 G for three minutes. Remove as much media as possible with minimal disturbance of the pellet as excess media can impact transfection efficiency.

Resuspend cells in 1.2 milliliters of the transfection buffer micro RNA plasmid mixture and set aside. Next resus, suspend the P life three prime UTR plasmid in transfection buffer in 96 well plates using a multi-channel pipette first transfer 32.4 microliters of transfection buffer into each well of a 96 well PCR plate. Then add 3.6 microliters of mini prepped P life, three prime UTR plasmid to each well and mix thoroughly.

Pipette 10 microliters of this mixture into each well of a 96 well transfection plate and cover it to avoid evaporation. Transfer 1.2 milliliters of the mixture of cells P life micro RNA plasmid and transfection buffer into a reservoir and mix well. Then add 10 microliters of this mixture into the first 96 well transfection plate already containing 10 microliters of transfection buffer plus P life.

Three prime UTR mix well by pipetting up and down several times. Placed in 96 well transfection plate on the cell electroporation device and initiate transfection. Once transfection is complete, add 100 microliters of prewarm media from a 96 well culture plate to each well of the 96 well transfection plate and mix.

Well move 100 microliters from each well into the 96 well culture plate Mix cells in the culture plate with the pipette positioned vertically in the center of the well to prevent cells from aggregating on the sides of the well. After all 3 96 well plates have been transfected culture cells for 48 to 72 hours at 37 degrees Celsius. To begin this procedure, prepare the lysis buffer by combining four parts water and one part five x passive lysis buffer in a reservoir.

Analyze each well for transfection efficiency using fluorescence microscopy. Identify wells with transfection efficiencies of 90%or higher wells that did not transfect efficiently or are expressing low levels of RFP indicative of overcrowding or media Exhaustion should be removed from the analysis completely. Remove the media from the cells being careful not to elude too quickly as it will cause cells to detach.

Add 26 microliters of lysis buffer to each well and place the plates on a plate shaker rocker at low moderate speed for 20 to 30 minutes while the cells are incubating in the lysis buffer. Prepare the two luciferase buffers for the dual luciferase assay. Wrap the two tubes for the firefly and ran vanilla buffers with aluminum foil as substrates may be light sensitive.

To prepare one x firefly luciferase buffer. First, add one milliliter of each of the first five 10 x firefly luciferase reagents, adding EGTA last to five milliliters of water to a final one X concentration. Next, add 0.205 grams of a TP to the 10 milliliter of one x firefly luciferase buffer.

Mix by inverting several times. Keep the a TP on ice at all times A TP will degrade. So if measuring more than one plate sequentially, the buffer must be made fresh beginning at this step for each additional plate.

Lastly, add 100 microliters of 100 x beta Lucifer, which is the substrate for the firefly luciferase protein. The buffer should change to a yellowish color based on its pH to reconstitute the one x vanilla luciferase buffer. Aliquot 10 milliliters of one X vanilla buffer.

For each 96 well plate and add 100 microliters of A BSA stock solution. Lastly, add 100 microliters of sealant ine to the buffer, adjust a pH of the one x farf fly buffer to eight and the one x vanilla buffer to five using sodium hydroxide and hydrogen chloride. Since the activity of each luciferase assay buffer is highly dependent on pH, it is important to accurately measure the pH.

To obtain consistent results, Bring the volume of each buffer to 10.5 milliliters with water to accommodate for luminometer priming. At this point, the cells should have been in the lysis buffer for about 20 minutes. Transfer the lysate to opaque white plates.

Use a multichannel pipette to remove 25 microliters from each well, and be sure to pipette up and down thoroughly to break up the clumps of cells and homogenize the lysate. Prepare the luminometer, turn it on, and select the protocol in the DLR folder named DLR with two injections. Select the wells to be tested.

Extend the delay before measurement setting to five seconds with a ten second integration time. Wash the capillaries, prime buffers once into the waist, and then prime a second time back into the buffer tubes. To ensure mixing, inject and prime the firefly and vanilla buffers in the left and right capillaries respectively.

Initiate the Lucifer assay. Each plate should take about 48 minutes to read After completion, save the file subsequently. Data analysis is performed as described in the accompanying protocol.

Text representative. Data produced with a three prime life assay is shown here. Each probe micro RNA is tested in quad duplicate.

The colors represent repression levels with red, indicating a strong micro RNA three prime UTR interaction. White boxes represent controls failed transfect or wells with low transfection efficiency. All replicates are averaged to produce high quality putative targets shown in the summary plate below the yellow arrows.

This table represents summary data of a subset of interactions produced using the three prime life spreadsheet. The repression index is used to call a putative micro RNA target with lower values corresponding to higher relative repression. The software calculates standard deviation, standard error, and Z-score for each interaction.

Statistically significant interactions are marked in red. In addition to comparing replicates with the normalized repression columns, the user can compare repression between the two microRNAs in the last four columns. This measure can indicate erroneous values from the cruciferous assay.

For example, abnormally high or low readings with a negative control, such as observed in row A nine, replicate number three. In addition, this measure can identify wells where the repression index may not indicate substantial repression, but that do exhibit significant differences between the microRNAs, such as in row C two In order to validate the microRNA targets identified by the three prime life assay. Other experiments such as micron, eye binding site deletion, northern blot or western blot can be performed.