title: ORCA3_8
organism: Catharanthus roseus
description: Protocols: Samples for further analysis were taken 3 weeks after inoculation of fresh plates and immediately flash frozen in liquid nitrogen. Hairy roots were initiated by infecting axenically grown C. roseus (Würzburg) leaves (15) with 2-day-old A. rhizogenes carrying the GUS or ORCA constructs. After incubation for 48 h on Murashige and Skoog medium containing 50 μM acetosyringone, the leaves were transferred to solid modified Gamborg B5 medium supplemented with 500 ppm cefotaxime (Duchefa) and 10 ppm meronem (Astra Zeneca) to eliminate Agrobacterium. Well-growing root clones were individually subcultured on the same medium without antibiotics. The transgenic nature of the resulting root clones and the absence of Agrobacterium were confirmed by PCR. Samples for further analysis were taken 3 weeks after inoculation of fresh plates and immediately flash frozen in liquid nitrogen. About 100 mg of frozen ground root material was used for RNA extraction using the Qiagen RNeasy mini kit. 450 μl Buffer RLT (supplemented with 10 μl of β-mercaptoethanol per ml Buffer RLT) was added and the mixture was vortexed vigorously. The lysate was transferred to a QIAshredder spin column placed in a 2 ml collection tube and centrifuged for 2 min at full speed. The supernatant was mixed with half a volume of ethanol and transferred to a an RNeasy spin column placed in a 2 ml collection tube followed by centrifugation for 15 seconds at full speed. 350 μl RW1 buffer was added and the column was centrifuged for 15s at max speed. Next an on column DNase digestion was performed and the column was washed again with 350 μl RW1 buffer. Next, the column was washed twice with 500 μl Buffer RPE. The RNA was eluated in a clean 1.5 mL eppendorf by adding 40 μl RNase-free water and centrifuging at maximum speed for 1 minute. Random primed cDNA library: Purification of poly-A containing mRNA molecules - mRNA fragmentation - Random primed cDNA synthesis - Adapter ligation and adapter specific PCR amplification