miR primer design
The tool for designing primers for miR-specific qPCR consist of the executable files miRprimer2.exe and miRprimer.exe and the source code files miRprimer2.rb, miRprimer2.py and miRprimer.rb including source code in Ruby 1.9.3.
miRprimer2.exe was build with OCRA (https://github.com/larsch/ocra) from miRprimer2.rb and miRprimer.exe was build with OCRA from miRprimer.rb.
miRprimer2.py was written in Python3 to perform (almost) as "miRprimer2.rb".
The tool was written for Windows and has been tested under Windows XP and Windows 7.
Use of the Ruby developer versions require that Ruby 1.9.3 or later is installed on the computer. Ruby can be downloaded from http://rubyinstaller.org/.
Automatic installation:
1) Download miRprimer_installer to a folder on your computer.
2) Double click on the the program icon and follow the instructions.
3) The installer will install miRprimer2.exe, the README file and the input file input_miRs.txt to the folder "C:/miRprimer2/".
Manual installation
1) Download miRprimer2.exe, miRprimer2.rb, miRprimer2.py, miRprimer.exe or miRprimer.rb to a folder on your computer.
2) Download the input file input_miRs.txt.
3) Optional: Download this manual called README to the same folder.
To design primers:
1) Place the miR sequences you want to design primers to in the file input_miRs.txt in the same folder as miRprimer2.
The sequences should be in strict fasta format:
>miR name
sequence
Please see the examples that are already in the file input_miRs.txt.
2) When the input is ready the primerdesign can be started by double clicking the miRprimer2 icon.
This will open a small dos window that will indicate the progress of the primer design.
3) Press enter to close the dos window when the design program has finished.
4) The result are stored in the file miR_primer_pairs.txt.
If the dos window does not show up or closes without requiring an enter it means that the microRNAs in the input file were not in the right fasta format. Please check the format of input_miRs.txt and try again.
Interpretation of the result:
Open the result file miR_primer_pairs.txt in MSexcel or OpenOffice calc.
The primer pair labeled best pair is the suggested pair.
However, if this primer pair does not perform well the program suggest other primer pairs that can be tested. For each primer pair the score of the pair and the scores of the forward primer and the reverse primer are reported. The pairs are ranked according to the expected performance, which unfortunately is not always the same as the performance in test.
Furthermore, miRprimer assigns a score for the difference between the primer pairs for each target calculated as a function of the difference in length of the miR-specific parts of the primers and the sequence of the last three nucleotides at the 3-end of the primers. Lower scores means that the primer pairs are more different and identical primer pairs have a score of 1.0.
These data can be found in the file result_comparison_of_pairs.txt.
Please cite:
Busk, PK. (2014). A tool for design of primers for microRNA-specific quantitative RT-qPCR. BMC Bioinformatics 15(1):29
if you use miRprimer.
Updates:
21.Feb.2014: Addition of miRprimer2.exe and miRprimer2.rb that puts more emphasis into avoiding primer pair combinations that can form primer dimers. Except for this new feature, the tools perform exactly as miRprimer.exe and miRprimer.rb.
2.Feb.2018: Addition of the Python3 implementation of miRprimer2: miRprimer2.py
Good luck!
Peter Busk, 2.Feb.2018
The software is provided as is, without warranty of any kind, express or implied, including but not limited to the warranties of merchantability, fitness for a particular purpose and noninfringement. In no event shall the authors or copyright holders be liable for any claim, damages or other liability, whether in an action of contract, tort or otherwise, arising from, out of or in connection with the software or the use or other dealings in the software.
Copyright 2013, 2014, 2015, 2016, 2017, 2018 Peter Kamp Busk
Licensed under the Apache License, Version 2.0 (the "License"); you may not use this file except in compliance with the License.
You may obtain a copy of the License at http://www.apache.org/licenses/LICENSE-2.0
Unless required by applicable law or agreed to in writing, software distributed under the License is distributed on an "AS IS" BASIS, WITHOUT WARRANTIES OR CONDITIONS OF ANY KIND, either express or implied. See the License for the specific language governing permissions and limitations under the License.
