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Method Article
Jinggui Fang
Jinggui Fang
Corresponding Author
License:
This work is licensed under a CC BY-NC 3.0 License. Read Full License
Computational prediction of microRNAs is one of the most important approaches in microRNA studies. However, validation of the predicted microRNAs’ precise sequences is essential for further studies on microRNA biogenesis, evolution and functions. Here, we report a highly efficient method of determining the precise sequences of computationally predicted miRNAs, which method combines a miRNA-enriched library preparation, two specific 3’ and 5’ miRNA RACE (miR-RACE) reactions, and sequence-directed cloning. miR-RACE has the potential to overcome the major disadvantage of computational miRNA prediction methods in that they can not predict the precise sequence of miRNAs, which could make the bioinformatic prediction of miRNAs more powerful and accurate. The efficiency of this method can be reflected from the precise sequence validation of the miRNAs computationally predicted in citrus, apple, and some other fruit crops. Our on-going research indicates that miR-RACE can also be very effective in the verification of sequences of some doubtful miRNAs obtained by deep sequencing of small RNA libraries. The protocol of miR-RACE is a rapid one to be executed and can be completed in 2-3 days.
Keywords
grape, microRNA, miR-RACE
Figure 1
Figure 2
Corresponding author: Jinggui Fang
E-mail: [email protected]
The authors declare no competing financial interests.
This is a list of supplementary files associated with this preprint. Click to download.
- supplement0.doc
Table_1 Example of the primers used for miR-5’ RACE, and miR-3’ RACEExample of the primers used for miR-5’ RACE, and miR-3’ RACE GSP1 is the specific primer for 5’ miR-RACE, and the underlined region base pairs with the 3’ poly (A)n; GSP2 is the specific primer used for 3’ miR-RACE, and the underlined region base pairs with the 5’ adaptor.
- supplement0.doc
Table 2 PROCEDURE step 3 PCR reaction system
- supplement0.doc
Table 4 PROCEDURE step 19 PCR reaction system
- supplement0.doc
Table 3 PROCEDURE step 11 PCR reaction system
- supplement0.doc
Table 5 PROCEDURE step 21 PCR reaction system
- supplement0.doc
Table 6 PROCEDURE step 26 PCR reaction system
- supplement0.doc
Table 7 PROCEDURE step 29 PCR reaction system
- supplement0.doc
Table 9 PROCEDURE step 34 PCR reaction system
- supplement0.doc
Table 8 PROCEDURE step 31 PCR reaction system
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Associated Publications
MiR-RACE, a New Efficient Approach to Determine the Precise Sequences of Computationally Identified Trifoliate Orange (Poncirus trifoliata) MicroRNAs
by Changnian Song, Jinggui Fang, Chen Wang, +4
PLoS ONE (30 November, 2010)
Computational identification of microRNAs in apple expressed sequence tags and validation of their precise sequences by miR-RACE
by Huaping Yu, Changnian Song, Qidong Jia, +5
An open repository of community-contributed protocols sponsored by Nature Research.
Learn more about Protocol Exchange
Method Article
Jinggui Fang
Jinggui Fang
Corresponding Author
Version History
CURRENT STATUS: Posted
Version 1
Posted 03 Dec, 2010
No community comments so far
Metrics
Comments: 0
HTML Views: ...
License:
This work is licensed under a CC BY-NC 3.0 License. Read Full License
Computational prediction of microRNAs is one of the most important approaches in microRNA studies. However, validation of the predicted microRNAs’ precise sequences is essential for further studies on microRNA biogenesis, evolution and functions. Here, we report a highly efficient method of determining the precise sequences of computationally predicted miRNAs, which method combines a miRNA-enriched library preparation, two specific 3’ and 5’ miRNA RACE (miR-RACE) reactions, and sequence-directed cloning. miR-RACE has the potential to overcome the major disadvantage of computational miRNA prediction methods in that they can not predict the precise sequence of miRNAs, which could make the bioinformatic prediction of miRNAs more powerful and accurate. The efficiency of this method can be reflected from the precise sequence validation of the miRNAs computationally predicted in citrus, apple, and some other fruit crops. Our on-going research indicates that miR-RACE can also be very effective in the verification of sequences of some doubtful miRNAs obtained by deep sequencing of small RNA libraries. The protocol of miR-RACE is a rapid one to be executed and can be completed in 2-3 days.
Figure 1
Figure 2
Corresponding author: Jinggui Fang
E-mail: [email protected]
The authors declare no competing financial interests.
This is a list of supplementary files associated with this preprint. Click to download.
- supplement0.doc
Table_1 Example of the primers used for miR-5’ RACE, and miR-3’ RACEExample of the primers used for miR-5’ RACE, and miR-3’ RACE GSP1 is the specific primer for 5’ miR-RACE, and the underlined region base pairs with the 3’ poly (A)n; GSP2 is the specific primer used for 3’ miR-RACE, and the underlined region base pairs with the 5’ adaptor.
- supplement0.doc
Table 2 PROCEDURE step 3 PCR reaction system
- supplement0.doc
Table 4 PROCEDURE step 19 PCR reaction system
- supplement0.doc
Table 3 PROCEDURE step 11 PCR reaction system
- supplement0.doc
Table 5 PROCEDURE step 21 PCR reaction system
- supplement0.doc
Table 6 PROCEDURE step 26 PCR reaction system
- supplement0.doc
Table 7 PROCEDURE step 29 PCR reaction system
- supplement0.doc
Table 9 PROCEDURE step 34 PCR reaction system
- supplement0.doc
Table 8 PROCEDURE step 31 PCR reaction system
Loading...
Associated Publications
MiR-RACE, a New Efficient Approach to Determine the Precise Sequences of Computationally Identified Trifoliate Orange (Poncirus trifoliata) MicroRNAs
by Changnian Song, Jinggui Fang, Chen Wang, +4
PLoS ONE (30 November, 2010)
Computational identification of microRNAs in apple expressed sequence tags and validation of their precise sequences by miR-RACE
by Huaping Yu, Changnian Song, Qidong Jia, +5