Synthesis of good quality double-stranded cDNA from the bark tissue of robusta coffee (Coffea canephora) plants

Quality RNA in large quantity is often required in the analysis of gene expression.RNA extraction from samples collected from woody plants is generally complex andbecomes the main limitation to study gene expression particularly in perennial crops likecoffee. Standard RNA extraction protocols are time consuming laborious and cannot beadapted for high throughput functional analysis. A simple and effective protocol forextraction of high quality total RNA from bark tissue of woody stem was achieved using theRNeasy plant mini kit (Qiagen, USA). The extracted RNA was successfully converted intodouble-stranded cDNA using the SMATer cDNA synthesis kit (Clontech, USA) which isbased on the Switching Mechanism At 5’ End of RNA Transcript (SMART) technology. Theintegrity of the total RNA used for synthesizing double stranded cDNA was assessed byamplifying a 1282 bp product targeting the glyceraldehyde 3-phospho dehydrogenase(GAPDH) gene by PCR. As expected, the PCR product contained the full coding sequenceplus 69 and 196 bp of 5’ and 3’ UTRs respectively. The double-stranded cDNA was usedsuccessfully for creating a SSH cDNA library (results not reported here). The cDNA couldalso be useful for a number of other applications like cDNA library construction, ESTanalysis, RACE and Next Generation Sequencing (NGS).