2. Material and Methods 1 Material
2.5. RNA Methods
2.5.1. RNA Isolation for northern blot and RT-PCR
Total RNA was extracted from Arabidopsis AT7 and tobacco BY-2 protoplasts, as well as from different tissues of Arabidopsis transgenic and wild-type plants using TriReagent (Molecular Research Center) according to the protocol suggested by the manufacturer. For 100 mg of plant tissue, 1 ml of Tri Reagent was added into a 2.0 ml screw-cap micro centrifuge tube. The sample was disrupted using a Tissue Lyser (Qiagen). The homogenized sample was incubated for 10 minutes at room temperature and then, 200 μl of chloroform were added followed 30 seconds of homogenization. After 10 minutes of incubation at room temperature, the sample was centrifuged at 12000g at 4°C for 10 minutes. The clear upper phase containing total RNA was collected to a new micro centrifuge tube containing 600 μl of isopropanol. The sample was incubated at room temperature for 5 minutes and centrifuged at 12000g at 4oC for 10 minutes. The supernatant was discarded and the pellet was washed with 70% ethanol. The RNA pellet was air dried for 10 minutes at room temperature and dissolved in DEPC-treated water. The total RNA was treated with DNase I according to the manufacturer’s protocol (Ambion), before any reverse transcription and northern blot experiments were performed. The RNA quality was analyzed by gel electrophoresis.
2.5.2. Formamide Gel
RNA samples (5μg of total RNA per lane) were mixed with 1 volume of sample buffer, which was previously denatured for 5 minutes at 65°C. Prior to loading the gel, 1/6 volume of loading buffer was added to each sample. RNA was separated on 1% agarose gel in MOPS-buffer with 2.2 M formaldehyde.
Sample buffer MOPS buffer Loading buffer
66% formamide 0.2M MOPS (pH 7.0) 50% Glycerol 8.6% formaldehyde 0.05M sodium acetate 1mM EDTA
2.5.3. cDNA synthesis
For reverse transcriptase reactions, Superscript reverse transcriptase II (Invitrogen) was used according to the manufacturer’s protocol. Reactions were done in 20 μl final volume with 2μg of RNA, 1mM oligo dT (dN-18T), 500μM of each dNTP, 1x First Strand Buffer (Invitrogen), 10μM of dithiothreitol and 20 units of reverse transcriptase II. For 5’RACE libraries (2.5.5), 10μg of adaptor ligated RNA (2.5.5.1) were used.
2.5.4. Small RNA northern blot
Northern blots to detect miRNAs were prepared from RNA samples of protoplasts transfected with a construct containing the precursor miRNA. The protocol for RNA polyacrylamide gel electrophoresis, transfer, probe labeling with radiochemical and hybridization were done following Llave et al., ( 2002).
2.5.4.1. RNA electrophoresis- polyacrilymide gel
RNA samples (20 μg of total RNA per lane) were mixed with one volume of urea loading buffer denatured for 4 minutes at 95°C and cooled down on ice for 5 minutes. The RNA was separated on 17% denaturating polyacrylamide gels in TBE buffer. Before loading the samples, a pre-run was done at 150 volts for 1 hour. After loading, the run was performed at 350 volts for 5 hours. An RNA oligonucleotide of 21 nt was used as a size marker. RNA was transferred to a nylon membrane (Hybond-N+, GE Biosciences) with Trans-blot SD Semi-dry Transfer Cell (Bio-Rad), at 400 mA, for one hour. The RNA was fixed in the membrane by UV crosslinking, with 1200μJ, followed by baking the membrane at 80oC for 30 minutes.
Polyacrylamide gel (DEPC water) Urea loading buffer (DEPC water)
7 M Urea 8 M Urea
0.5X TBE buffer 0.05 % (w/v) Bromophenol blue 0.5 mM TEMED 0.05 % (w/v) Xylencyanol 2 mM Ammonium Persulfate 0.5 mM EDTA
10x TBE buffer (DEPC water)
0.9 M Tris 0.9 M Boric Acid 0.02 M EDTA
2.5.4.2. Preparation of Radiolabelled DNA probe
Hybridization probes were prepared with 20 μM oligonuclotides, whose sequences were complementary to investigated miRNAs. Probes were labeled with [32P] γ- ATP (5000ci/mmol; 10 mCi/ml, from Hartmann Analytic GmBH, Germany) using polynucleotide kinase (New England Biolabs). The labeled probes were purified with Sephadex G25 spin columns (GE Biosciences).
2.5.4.3. Hybridization
RNA blots were pre-hybridized for 30 minutes at 42°C in PerfectHyb™Plus (Sigma) hybridization buffer. After adding the probe, hybridization was carried out overnight at 42°C. After hybridization, membranes were washed with decreasing concentrations of SSC solution containing SDS (2X SSC, 0.2% SDS; 1X SSC, 0.2% SDS; 1X SSC, 0.1% SDS for 20 minutes each wash with rotation at 50°C). Dried membranes were exposed to Phosphoimaging plates (Kodak), which were read out in a Typhoon scanner (Amersham- GE Biosciences).
2.5.5. 5’RACE
2.5.5.1. RNA adaptor ligation
The validation of miRNA targets takes advantage of a modified RNA ligase- mediated Rapid Amplification of cDNAs Ends (5´RACE) approach, which can be used to precisely map the position of the cleavage induced by the RISC complex (Llave et al., 2002a). To construct a 5´RACE library for every target, total RNA was isolated from AT7 protoplasts co-transfected with plasmids that enables the protoplasts to express both the miRNA precursor and the target cDNA in translational fusion with GFP. An RNA adaptor (300ng) was ligated to 10 μg of total RNA using T4 RNA ligase (New England Biolabs) in 1x ligation buffer at 37oC for 1 hour. The synthesis of cDNA was described in 2.5.3.
2.5.5.2. Nested PCR
The RNA adaptor provides an anchoring sequence for PCR primers. A nested PCR with the outer 5RACE primer and a gene specific primer 1 (GSP1) was performed. The nesting reaction was performed with the Inner 5RACE primer and a GSP2 primer. PCR products were gel analyzed and cloned with TOPO-TA cloning kit (Invitrogen). Alternatively, PCR fragments were gel purified with Qiaquick Gel Purification Kit (Qiagen) and then cloned with TOPO-TA cloning kit (Invitrogen). Positive clones were screened using colony PCR (2.4.2.2). In this case another gene specific primer (GSP3) was used with the Inner 5RACE primer. Between 5 and 10 were sequenced clones for each target. Cycling conditions for nested and nesting PCR were done in such a way to optimize the yield and specificity. Therefore, for these reactions, the Hot Start PCR approach was applied. In addition, the touchdown PCR (TD-PCR) approach was also implemented in these reactions. In TD-PCR, the annealing temperature is set 10 degrees higher than in normal PCR and, after each cycle (denaturation, annealing and extension), the annealing temperature decreases by one degree per cycle (ten cycles). Then, the annealing temperature of the tenth cycle is maintained through the rest of 25 cycles. The set up of these PCRs is the same as presented for the Hot Start PCR (2.4.2.4). However, for the nested reaction, 1μl of cDNA was added and in the nesting reactions, between 0.5 to 5μl of nested PCR was added.