title: Triticum aestivum; 454 Titanium; normalised cDNA from seedling leaves in a circadian time-course
design: Total RNA was extracted using the RNeasy mini prep kit (Qiagen), and tri-reagent (Sigma) following the protocol online at www.mrcgene.com/tri/htm. RNA was extracted from seeds using protocol 2 from Onate-Sanchez, L. and Vicente-Carbajosa, J. "DNA-free RNA isolation protocols for Arabidopsis thaliana, including seeds and siliques." BMC Res Notes 1, 93, (2008). Between 0.5g and 1g of frozen material ground in liquid nitrogen with a cold mortar and pestle and extracted. RNA was treated with DNase (Roche), followed by 0.8ug/ul protease K (Roche, 20ug/ul stock), extracted by phenol/chloroform and precipitated with addition 1/10th v/v 3M sodium acetate pH 5.2, 1/1000th v/v glycogen (Roche, 20ug/ul stock) and 3 volumes of ethanol. RNA was subsequently purified using RNeasy MINElute clean up kit (Qiagen) and analysed with an Agilent Bioanalyser.  mRNA was isolated using an Oligotex mRNA mini kit (Qiagen), using up to 250ug total RNA per column. cDNA was synthesizing from either 0.5ug mRNA or 3ug total RNA pools. First strand cDNA synthesis was performed using the reagents from the cDNA MINT-Universal kit (Evrogen), but using custom primers that carry a 1 base modification creating an MmeI site. Two microliters of 1st strand cDNA was amplified by PCR using MmeI M1 primer: 5’AAGCAGTGGTATCCAACGCAGAGT 3’. Thermal cycler parameters were 95oC 1 min followed by 10-15 cycles of 95oC for 15 secs, 63oC for 30 secs 72oC for 3 mins, ending with 1 cycle of 72oC for 10 mins. PCR was performed using the Advantage 2 PCR kit (Clontech). After amplification cDNA was purified on a Qiagen Minielute column and eluted in 30 ul EB. Purified cDNA (120ng) was used in the normalization procedure as described in the Evrogen TRIMMER kit.