Parallel analysis of 5''-RNA ends in mock inoculated tomato plants

5' RNA ligase mediated rapid amplification of cDNA ends (5' RLM-RACE) is the widely used tool for confirming the direct cleavage of target mRNA by a miRNA. Recently, 5' RLM-RACE combined with high-throughput sequencing such as parallel analysis of RNA ends (PARE) are gaining importance as these techniques allows to assess the large number of sample in a single experiment. In order to verify the cleavage of predicted a target mRNA in potato spindle tuber viroid (PSTVd) infected tomato plants, the the leaf samples collected from the mock inoculated (control/healthy) at 21-days were subjected for RNA extraction. A custom RNA adaptor containing MmeI restriction endonuclease was ligated to the free 5''-phosphate end of an uncapped mRNA using T4 RNA ligase, followed by reverse transcription (RT) and second strand synthesis using poly (T) primers. Obtained product was slightly amplified, cleaved with MmeI restriction endonuclease and then, a dsDNA adaptor was added to the 3’-end of the MmeI restriction endonuclease cleavage site. Resulted products were sequence in an Illumina Mi-Seq machine. Retrieved data was then used to verify the 5’-termini of predicted target mRNA. Overall design: In order to verify the PSTVd mediated cleavage of mRNAs, the PARE data obtained from control plants were compared with the PARE data obtained with PSTVd infected plants.