RNA editing play a critical role in human disease. Scientist are finding endless application in the medicines. This protocol demonstrate the application of portable temperature electrophoresis technology as a non sequencing approach for the rapid and reliable identification of RNA modification in RNA editing without the need of direct RNA sequencing.
The micro tTG electrophoresis based approach was used to characterize the differences between the melting profile of four edited RNA fragments and their corresponding non-edited fragments. The pattern similarity scores were used to assess the reproducibility of the method. This platform enable the detection of even single-based substitution in RNAs, in straightforward, simple, and cost effective manner.
It is anticipated that this analytical tool will aid new finding in molecular biology Begin by identifying gene fragments with different melting profiles and generate predicted melting curves using the Umelt HETS web-based tool. Design three pairs of 300 to 324 base pair gene fragments for each target gene. Select the non-edited or edited pair that shows the maximum difference between the melting regions on the helicity axis for further analysis.
For micro-TGGE analysis, synthesize the selected gene fragment by PCR amplification. Design the forward and reverse primers using the DNA dynamo software, and verify the primer sequence using the NCBI Primer-BLAST tool. Extract total RNA from the source of edited and non-edited genes using standard methods.
Synthesize the corresponding cDNA, prepare 20 microliters of the PCR reaction mixture, and set up the PCR as described in the text manuscript. Then, mix six microliters of the PCR product with three microliters of 6x gel loading dye in a 250 microliter tube, and make up the total volume to 12 microliters with sterile water. Store the PCR product at 25 degrees Celsius until further use.
To assemble the gel cassettes, sandwich the top gel plate between the other two plates in the gel cassette holder for gel polymerization. To prepare the poly acrylamide gel, add 7.2 grams of urea to a 50 milliliter tube and dissolve in 10 milliliters of sterile water. Heat the sample in a microwave for 20 to 30 seconds.
And let it cool to room temperature. Add three milliliters of 5x TBE buffer, 2.25 milliliters of acrylamide bis, 75 microliters of 10x ammonium persulfate, and 15 microliters of TEMED to the solution. Pour the gel solution into the gel cassette holder slowly at a tilted angle to avoid air bubbles.
After around 30 minutes, disassemble the gel cassettes and clean them. Use the micro-TGGE apparatus with a temperature gradient set perpendicular to the direction of DNA migration. Soak the upper and lower electrophoresis buffer pads in two milliliters of 1x TBE buffer.
Place the gel cassette in the horizontal electrophoresis chamber unit, and position the upper and lower buffer pads. Then load 10 microliters of the PCR product into the middle well, and one microliter of the PCR product into each side well. Wait for a minute, connect the power unit and supply 100 volts for 12 minutes at a linear temperature gradient of 15 to 65 degrees Celsius.
After the run has completed. take out the cassette and remove the upper glass cover. Pour 300 microliters of 10x SYBR Gold stain onto the gel.
Visualize the melting profiles using the blue LED flashlight installed in the palm-sized electrophoretic device. Repeat every electrophoretic experiment thrice to confirm the reproducibility of the data. Download and open the micro-TGGE analyzer software.
Open the JPEG file containing the gel image. Click on the frame"button and select the appropriate frame of the gel image. Click on the coordinate correction"button and add two reference points.
Click on the addition of feature points"button and add sample points. Then, save the processed gel image data in micro-TGGE format. Click on the sample button and select the search for simple points"option to compare two or more images.
For the C-to-U RNA editing type, the non-edited sample with the original cBase, showed a longer melting pattern at the strand end melting point than the edited sample with the modified U base. For the A-to-I RNA editing type, the edited sample with the modified G base, displayed a longer melting pattern at the strand-end melting point than the non-edited sample with the original A base. For the reverse U-to-C RNA editing type, the edited sample in the first gene with the modified C base showed a longer melting pattern between the strand's initial and end melting points than the non-edited sample with the original U base.
However, a similar pattern was not observed for the other gene. The pattern similarity scores of the C-to-U and A-to-I RNA editing types were lower than those of the two U-to-C RNA editing types. This difference was likely related to the respective locations of the editing sites.
The uMelt HETS analysis showed the C-to-U modification would shift the melting curve to the left along the temperature axis. The pattern similarity scores calculated from micro-TGGE analyses of the non-edited and the three edited fragments were consistent with the results predicted using uMelt. Optimization of the target gene fragment is critical for clear differentiation between the edited and non-edited reason.
And this process has been simplified in the current protocol.
