Abstract
Using a Reverse Transcriptase-PCR approach spliced transcripts can be converted to cDNA, amplified and cloned into an expression plasmid. Sequencing of the obtained cDNA allows identification of the splicing events that generated the detected RNA (Grewe et al., 2012).
Keywords: Pre-mRNA, Splicing, Transcription, RT-PCR, Retrovirus
Materials and Reagents
I. RT-PCR
II. Cloning
Equipment
Procedure
Depending on the experiment isolation of cytoplasmic or total RNA should be done. In general, cytoplasmic RNA is enriched in spliced RNA lacking all introns whereas total RNA contains unspliced, partially-spliced and fully-spliced RNA. A protocol for the isolation of cytoplasmic RNA from mammalian cells can be obtained from the first part of the bio-protocol "Packaging of retroviral RNA into viral particles analyzed by quantitative Reverse Transcriptase-PCR". Sense and antisense primers should hybridize upstream of the splice donor and downstream of the splice acceptor sequence, respectively (Figure 1A). It is also possible that one primer overlaps the exon-exon junction ensuring perfect hybridization to spliced RNA only. For subsequent cloning, both primers should contain recognition sites for restriction endonucleases (cloning via restriction enzymes) or sequences identical to the acceptor plasmid (cloning via homologous sequences) at their 5' ends. A cDNA containing the whole open reading frame (ORF) of a gene can be generated when sense and antisense primers hybridize to the beginning of the ORF sequence or to the 5' untranslated region and to the end of the ORF sequence or the 3' untranslated region, respectively (Figure 1B).
Recipes
Acknowledgments
The protocol was adapted from our paper Grewe et al. (2012). This work was funded by a grant from the German Research Foundation (DFG) to Klaus Überla (Ue45/11-1). Bianca Hoffmann is and Bastian Grewe was supported by a fellowship from the DFG graduate school (GRK 1045). Beside Bianca Hoffmann and Bastian Grewe, Katrin Ehrhardt, Maik Blissenbach, Sabine Brandt, Klaus Überla, Alexander Stang, Thomas Grunwald, Klaus Sure, and Bettina Tippler were part of the team which established the methods described.
References
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