The overall goal of this procedure is to successfully examine three prime mRNA cleavage in vitro. This is accomplished by first making transcriptionally active nuclear extract from cells grown in suspension. The second step of the procedure is to make radio labeled RNA substrates via in vitro transcription.
The third step is to gel purify the labeled RNA substrates. The final step is to assemble the cleavage reaction, including the nuclear extract and labeled RNA substrate. Ultimately, results can show three prime mRNA, UNC cleaved substrates, and cleaved product through vertical radioactive gel electrophoresis.
Visual Demonstration of this method is important, even though it has been performed since the 1980s, it is not easily executed as there are many specific steps throughout the procedure that are challenging. Have washed cells, pelleted, and ready for lysing. To begin this protocol to the cell pellet, add buffer with fresh DTT.
Add five volumes of the estimated CPV and use a pipette to break up the pellet and allow the suspension to chill on ice. After 10 minutes, spin the cells down at four degrees Celsius in a swinging bucket. Centrifuge carefully aspirate off the supernatant.
The pellet will have absorbed some solution and swelled in size. Then add one CPV of buffer A, and gently break up the pellet with a pipette tip. Pour the suspension into a chilled down homogenizer, and with five to 12 strokes of a tight pestle ly the cells.
Remove a small aliquot, apply trian blue and check the cells to determine the completeness of their lysis. If needed, continue homogenizing the cells until they are 90%lysed, but don't overdo it. Now transfer the lysate to an ultra clear ultracentrifuge tube.
Record the total volume and spin it as done previously. Aspirate the cloudy supernatant and save it on ice. And for cytosol analysis, be careful to not disturb the pellet.
It contains the nuclei. Next, spin down the pellet at high speed for 15 minutes. Remove the small volume of remaining supernatant and pool it with the cytosol collection.
Then determine the PACT nuclear volume. Add a volume equivalent of buffer C to the pellet. Then dislodge the pellet using the pipette in a clean down homogenizer.
Use five to 10 tight pestle strokes to resus. Suspend the pellet. Transfer the homogenate to a chilled tube and mix it by inversion for 30 minutes.
At four degrees Celsius, a stir plate may be used. If the volume is large enough, transfer the lysate to an UltraClear ultracentrifuge tube and at four degrees Celsius, spin it down for 30 minutes. At about 25, 000 gs, the supernatant is the nuclear extract.
Following the text protocol, complete the dialysis from just one microgram of template. Prepare the probe by in vitro transcription using a kit with an SP six or T seven compatible promoter. When loading the page, use three microliters of marker and all 25 microliters of the reaction for each RNA substrate.
Also prevent cross-contamination by spacing out the lanes of similarly sized transcripts. Run the gel such that a 600 nucleotide probe would run at 25 watts for about two hours later. To mark the location of gel bands, illuminate the exposed film with a light box and put the wrapped gel on top.
Align the auto rad marker and the exposed marker on the film. And then mark the band locations with black marker to remove the bands Unwrap the gel. And with a clean blade excise the band sections of the gel transfer each section to its own tube and elute out the RNA following standard procedures.
After all reagents have been prepared, heat the labeled RNA to 80 degrees Celsius for two minutes. Then transfer the RNA to ice vortex, the 10%PVA, and then spin it down at high speed for a few minutes to remove bubbles, then proceed to make the master mixes for each reaction. For each reaction, dispense 5.25 microliters of master mix one.
Next, add 6.25 microliters of the nuclear extract mix. Then add a microliter of labeled RNA substrate diluted to 50 ano molar in all. Each reaction uses less than five ano molar labeled RNA.
Now, gently vortex and quickly spin down the reaction mixes. Allow the reaction to take place for up to two hours at 30 degrees Celsius. Taking a time course of the reaction will provide the best results After using proteinase K to digest the protein in the reaction and then purifying the reaction Using the phenol chloroform method, run the products out on a 6%page load, three microliters of marker or five microliters per reaction product per lane.
Then run the gel and later carefully disassemble the gel apparatus. To collect the gel, press filter paper firmly against it and flip it over. Firmly press the glass to ensure the gel is fully gripped by the paper.
Then slowly peel the filter paper off with the gel attached. Wrap the exposed side of the gel in plastic and tape the wrap to the filter paper on the other side. Finish by putting the gel in a dryer for 15 minutes with the filter paper facing the vacuum.
Later, analyze the gel by exposing it to a film or phospho imager. The cleaved. RNA substrate is the slowest migrating band at the top of the gel.
The specific cleaved product marked by a bold arrow, runs faster in the gel at the expected size. This product is absent from the control with a point mutation in the pollier sequence. Quantification of the cleavage activity was determined with a DS metric plot.
Comparing the ratio of cleaved to cleaved, RNA, normalized to the cleaved RNA control. Following this procedure, other methods like a polyadenylation assay can be performed to answer additional questions such as the activity of polyA polymerase. This assay is done by altering the reaction conditions such as adding magnesium and removing EDTA.
