cDNA Reverse-transcription
To remove contaminating DNA, It is recommended that RNA be treated with DNase using the DNA Free kit (Ambion). This involves incubating 10 mg of RNA at 37°C in a 50 ml volume reaction, containin of 2U rDNaseI, 5 m l 10x DNase I buffer for 30 minutes. Following incubation 5 ml of DNase inactivation reagent is added to the RNA, and mixed by pipette. Following a 90 second spin at 10000 x g in a microcentrifuge, the supernatant, which contains the RNA is transferred to a fresh Eppendorf.
Construction of cDNA Using Reverse Transcriptase
Superscript II Reverse transcriptase (Invitrogen) is used to construct cDNA. ~2 mg of DNA free treated RNA is mixed with 1 ml Oligo d(T) in an Eppendorf tube, and incubated at 70°C for 10 minutes. The Eppendorf tube is then transferred to ice and the following components added, 4 ml 1st strand synthesis buffer, 2 ml 0.1M DTT, 1 ml 10mM dNTPs, 1 ml RNase inhibitor, 1 ml Superscript II reverse transcriptase. The reaction is made up to 20 ml in nuclease free H20. The mixture is then incubated at 42°C for 45 minutes to allow the reverse transcription reaction to occur. The tube is then transferred to a 75°C water bath for 15 minutes, to destroy the enzymes. The tube is span down, and further diluted with the addition of 80 ml of nuclease free water. 10 ml of the product is used for subsequent PCR reactions.
Microarray Analysis Filtering
RNA was harvested using the Qiagen RNeasy Plant mini kit extraction protocol. 10 mg of total RNA was sent for hybridisation to Affymetrix chips. Data received from the facilities was analysed using the Affymetrix microarray software suite. Initially GeneChip data was normalised and scaled to a value of 500. Following comparison of experiment to baseline GeneChips, filtering analysis was carried out to identify differentially regulated genes.
Firstly detection values were filtered: all probe Ids whose detection was deemed significantly absent by the software, or had a signal level <50 were eliminated from the data set. The second microarray analysis filter eliminated all probe Ids that were deemed to have no significant change in expression by the software. Finally all probe Ids with a SLR between –0.999 and 0.999 were eliminated. The datasets obtained were exported to Microsoft excel and compared to update annotation data available from the Sheen and Schroeder labs using Microsoft Access (http://genetics.mgh.Harvard.edu/sheenlab; www.biology.ucsd.edu/labs/schroeder/genechips.htlm).
This resulted in statistical significant datasets containing genes differentially regulated to a SLR ≥ 1 or ≤ -1 with correct TIGR annotations.
GUS reporter Activity, xgluc
Freshly harvested whole seedlings or leaf tissue is incubated in a multi-well polypropylene plate containing absolute heptane for 10 minutes at ambient room temperature. Following removal of the heptane, plant tissue is air dried for 5 min in a flow hood.
Plants are then immersed in GUS solution (1mM X-Gluc (Roth), 0.5mM potassium ferrocyanide, 0.1% (v/v) triton X-100, 100mM sodium phosphate buffer pH 7.0), and incubated at 37°C overnight. The following day the X-Gluc solution should be discarded. The tissue is washed repeatedly with 70% ethanol to remove chlorophyll. This enables the GUS stain to be visualised.
Seed Sterilisation and Plant Growth
Bleach seed sterilization
To perform bleach seed sterilisation, firstly seeds should either be wrapped in a miracloth package (Cal-Biochem) or placed in an Eppendorf tube. Seeds are then immersed in 70% ethanol for two minutes, followed by a 5 minute soak in 5% bleach 0.5% SDS. Seeds should then be rinsed six times with sterile water. Seeds in te miracloth are air dried in the fume hood, and sprinkled onto GM plates; seeds in Eppendorf tubes can be pipetted onto GM plates.
Chlorine Gas Seed Sterilisation
When performing sterilization by using chlorine gas, firstly seeds in open Eppendorf tubes should be placed into a desiccator jar in a fume hood. A beaker containing 100ml of bleach is then placed in the desiccator next to the seeds. 3ml of concentrated HCL is added to the beaker to create chlorine gas. The desiccator jar should be quickly sealed and a vacuum applied. Seeds require to be exposed to the chlorine gas for six and a half hours. Following release of the gas, the tubes should be quickly sealed and transferred to a laminar flow hood, and transferred to GM growth plates.
Following transfer of seeds to GM plates, containing the relevant antibiotics, plates should be sealed with Micropore surgical tape (3M), then covered with tin foil and stored at 4°C for 3-5 days to imbibe seeds. After this period the Tin foil wrap can then be removed from the plates, and the plates transferred to Percival growth cabinets with the relevant growth conditions.