Title: Affymetrix CEL analysis. Description:
Title: Affymetrix CHP Analysis (ExpressionCall). Description:
Lotus japonicus (Regel) Larsen cv. Gifu was initially obtained from Professor Jens Stougaard (Aarhus University, Aarhus, Denmark) and then self-propagated at the University of Seville. The Ljgln2-2 mutant was isolated from photorespiratory mutant screening. The mutant progeny of two consecutive backcrosses with the wild type background (WT) were used. WT and mutant seeds were scarified and surface-sterilized, germinated in 1% agar Petri dishes, and transferred to pots using vermiculite as solid support. Five seedlings were planted in each pot and grown during 45 d in a growth chamber under 16 h : 8 h day : night, 20 : 18C, with a photosynthetic photon flux density of 250 umol m-2 s-1 and a constant humidity of 70%. CO2 was automatically injected to a final concentration of 0.7% (v/v) to allow for normal growth of the Ljgln2-2 mutant in a photorespiration-suppressed atmosphere. Plants were watered with Hornum nutrient solution, containing 5 mM NH4NO3 and 3 mM KNO3. After 45 days of growth under CO2-enriched atmosphere, leaf tissue was harvested for each plant genotype, constituting the time zero point. The plants were then transferred to normal CO2 conditions (0.04%) and, at different time points, each plant genotype was sampled. Every harvest involved at least three independent biological replicates for each genotype/time point. A biological replicate consisted of tissue pooled from five plants grown in the same pot.(Parameters: time unit = seconds, min temperature = 18, max temperature = 20, temperature unit = C, media = Hornum)
Total RNA was isolated using a hot borate method: Buffer: 200mM Na-Borate (Sodium Tetraborate Decahydrate); pH 9, 30 mM EGTA 5 mM EDTA 1 % SDS 1 % Na-Deoxycholate Add fresh: 10 mM DTT (100ul / 5ml buffer; 0.5M) 100 mM ß-Mercaptoethanol (35 ul / 5 ml buffer, 14.3 M Stock solution) 2 % PVPP 2 % PVP (PVP40) Procedure: 1. Grinding Homogenise frozen sample. Heat the Borate-Buffer (incl. DTT, PVPP, PVP, ß-Merc) to 95C. Add 1ml hot buffer to the sample (100-150 mg) and mix extensively by vortexing. 2. Protein digest Add 0.15 mg Proteinase K (spatula tip), mix and incubate for 45-60 min at 37C on a thermo mixer (slightly shaking). Add 78 ul 2 M KCl / ml, mix and incubate at least for 30 min on ice. Spin down the homogenisate for 10 min at 12000 rpm and transfer the supernatant into a new 2 ml eppendorf cup, discard the pellet. 3. Extraction Add 1 volume ice-cold Chloroform / Isoamylalcohol (-20C) invert tube; spin for 5 min at 14000 rpm. Transfer the upper (aqueous) phase into a new eppendorf tube. Add ½ Vol cold Phenol (acidic pH) and mix the sample. Add ½ Vol cold Chloroform / Isoamylalcohol (24:1) and mix again. Centrifuge for 5 min at 14000 rpm and transfer the supernatant into a new eppendorf tube. Repeat the extraction 3 times. Mix aqueous phase with 1 volume ice-cold chloroform and spin down at 14000 rpm for 5 min. Transfer the obtained aqueous layer into a fresh 2 ml eppendorf tube. 4. Isopropanol precipitation Add 1 Vol ice-cold (-20C) Isopropanol, invert tube gently several times and incubate on ice at least 30 min. Spin down at 14000 rpm for 10 min and discard the supernatant. Resuspend the pellet in 200-700ul DEPC-H2O on ice. Spin down at 14000 rpm and transfer into 1.5 ml eppendorf tube. 5. LiCl- precipitation Add 1 Vol 4M LiCl and incubate overnight at 4C in ice. 6. Wash with 70 % Ethanol Spin down for 10 min at14000 rpm and discard supernatant. Wash pellet with 2M LiCl (150-300ul) and spin for 10 min at 14000 rpm. Wash pellet 2 times with ice-cold (-20C) 70 % Ethanol and incubate the eppendorf cup on ice for 20 min. Centrifuge for 10 min at 14000 rpm and remove ethanol. 7. RNA- Resuspension Dry pellet at room temperature and resuspend in 10-50 ul of RNAse free water (Sigma). If necessary, RNA was further purified with the RNeasy spin columns (Quiagen). The quality and quantity of RNA were assessed with a Bioanalyzer-2100 using RNA 6000 NanoChips (Agilent Technologies) and a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies), respectively. (Parameters: Extracted product = total_RNA, Amplification = none)
Al the leaves from each biological replicate were harvested in situ and immediately frozen in liquid nitrogen. Samples from WT and mutant were taken at the same moment, namely after four hours from the beginning of the light period. Each sample was grinded with cooled mortar and pestle and stored at -80C untill needed.