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Updated: Jul 2, 2026
A Seamless Cloning Approach for Porcine Reproductive and Respiratory Syndrome Virus Expression Vector Construction
Published on: May 17, 2024
Concatameric cloning of porcine microRNA molecules after assembly PCR
Soroush Sharbati-Tehrani1, Barbara Kutz-Lohroff, Jutta Scholven
1Institute of Veterinary-Biochemistry, Freie Universität Berlin, Oertzenweg 19b, 14163 Berlin, Germany.
Abstract:
While the number of human or murine microRNAs (miRNAs) increases continuously, there are limited data available from other species. We report a novel identification method of small RNAs such as miRNAs, which allows simultaneous cloning of five RNA molecules within the same insert. First, RNA molecules <40nt were polyadenylated and five concatamerising 5' DNA adaptors were ligated to the molecules in independent reactions. Reverse transcription was carried out using oligo d(T)(18) primers with concatamerising 5' overhangs. The introduced complementary termini in the different reactions enabled the subsequent coupling of five purified antisense strands to one molecule by means of an assembly PCR. After cloning, small RNAs were identified by DNA sequencing. By means of this cloning approach, we identified 10 novel and one known porcine miRNAs. Furthermore, the endogenous expression of the cloned miRNAs was quantified in various tissues using a qRT-PCR approach.
Insights
Researchers developed a new method to identify small RNAs, like microRNAs (miRNAs), in species other than humans or mice. This technique successfully identified 10 new porcine miRNAs, expanding our understanding of miRNA diversity.
Area of Science:
- Biochemistry
- Genomics
- Molecular Biology
Background:
- Limited microRNA (miRNA) data exists for non-human, non-murine species.
- Advancements in sequencing technologies necessitate novel methods for small RNA identification.
Purpose of the Study:
- To develop and validate a novel method for identifying small RNAs, including miRNAs, from various species.
- To discover novel porcine miRNAs and analyze their expression patterns.
Main Methods:
- A novel method enabling simultaneous cloning of five small RNA molecules (<40nt) per insert.
- Polyadenylation of small RNAs, ligation of concatamerizing 5' DNA adaptors, and reverse transcription using oligo d(T)(18) primers.
- Assembly PCR for coupling antisense strands, followed by DNA sequencing for small RNA identification and qRT-PCR for expression analysis.
Main Results:
- Identification of 10 novel and 1 known porcine microRNA.
- Successful application of the novel cloning approach for small RNA discovery in a non-model organism.
- Quantification of endogenous miRNA expression in various porcine tissues using qRT-PCR.
Conclusions:
- The developed method is effective for discovering novel miRNAs in underrepresented species.
- This study significantly expands the known repertoire of porcine miRNAs.
- The findings provide a foundation for further research into miRNA function and regulation in pigs.
