ena-STUDY-Finnish Food Safety Authority Evira-16-04-2018-13:34:44:367-192
Deep sequencing of small RNA fraction isolated from strawberry, raspberry and Ribes sp., and from tobacco plants infected with different infectious clones of Potato virus A and Potato virus Y.
Total RNA was isolated from leaves of cultivated strawberries (Fragaria × ananassa), raspberries (Rubus idaeus L.) and Ribes species obtained from the genetic resource collection maintained by the Natural Resources Institute of Finland (LUKE). Total RNA was also extreacted from tobacco plants (Nicotiana tabacum L. cv. Samsun nn)inoculated with infectious clones of Potato virus A (PVA) engineered to express red fluorescent protein (PVA-RFP), green fluorescent protein (PVA-GFP) or ß-glucuronidase (PVA-GUS) and an infectious clone of Potato virus Y (PVY). Amount of viral RNA in the tobacco plants was estimated by quantitative PCR using RNA extracted from viral particles to draw the standard curve. RNA extracted from the berries was pooled into four unique pools and different amounts of viral RNA (PVY, PVA-RFP, PVA-GFP or PVA-GUS) were added to the four RNA pools. RNAs shorter than 30 nucleotides (nt) were isolated from the gel following acrylamide gel electrophoresis. Libraries for sequencing were prepared using the TruSeq small RNA kit (Illumina). Sequencing was carried out with an Illumina HiSeq 2500 instrument using the 50-bp single-end mode.
Total RNA was isolated from leaves of cultivated strawberries (Fragaria × ananassa), raspberries (Rubus idaeus L.) and Ribes species obtained from the genetic resource collection maintained by the Natural Resources Institute of Finland (LUKE). Total RNA was also extreacted from tobacco plants (Nicotiana tabacum L. cv. Samsun nn)inoculated with infectious clones of Potato virus A (PVA) engineered to express red fluorescent protein (PVA-RFP), green fluorescent protein (PVA-GFP) or ß-glucuronidase (PVA-GUS) and an infectious clone of Potato virus Y (PVY). Amount of viral RNA in the tobacco plants was estimated by quantitative PCR using RNA extracted from viral particles to draw the standard curve. RNA extracted from the berries was pooled into four unique pools and different amounts of viral RNA (PVY, PVA-RFP, PVA-GFP or PVA-GUS) were added to the four RNA pools. RNAs shorter than 30 nucleotides (nt) were isolated from the gel following acrylamide gel electrophoresis. Libraries for sequencing were prepared using the TruSeq small RNA kit (Illumina). Sequencing was carried out with an Illumina HiSeq 2500 instrument using the 50-bp single-end mode.