title: UH-UGA Rice PM 15.5k cDNA Array
status: Public on Nov 12 2009
contact.city: Hyderabad
contact.first: Markandeya
contact.last: Gorantla
contact.email: gorantla99_eprpharma@yahoo.com
contact.state: Andhra Pradesh
contact.address: University of Hyderabad
contact.department: Department of Plant Science
contact.country: India
contact.institute: university of Hyderabad
contact.phone: +919866023556
organism: Oryza sativa
manufacture_protocol: 1. cDNA microarray chip consisting of 15552 features with 5664 ESTs from drought stress induced cDNA library of Oryza sativa cv Nagina22,  1152 ESTs from drought stressed Pennisetum glaucum,  24 yeast artificial genes  and  48 samples of λ (Lambda) DNA, (Takara, Japan).  cDNAs  were printed in duplicate on superamine coated glass substrate using Omnigrid (GeneMachines).  The machine was calibrated for 384 Nunc square well plates, CMP3 print head and stealth printing pins and 75mm x 25 mm microarray glass slides were used, we have used 16 pins altogether in 4 x 4 format, which will give us an option to print 4 subarrays along Y direction and 4 arrays in X direction. The origin offset values set were 3000 micron in X direction and 2000 microns in Y direction. We have used 225μ spot to spot spacing in a subarray. The No of spots printed in a subarray is 18 clones in X direction and 18 clones in Y direction, the number of clones printed in one su
  barray is 18 x 18 = 324 stamps, in which 162 clones have been printed onto each subarray. The maximum number of arrays which would be printed were 4 in which we limited to 3 arrays, with a spacing of 500 microns each, in which a maximum number of 7776 clones in duplicate could be printed. The above format allowed us to print 7680 samples in duplicate including blanks. For printing the sample intake was for every 100 slides, the dip time being 1000 milliseconds, with each dip the sample is taken in by capillary action and is sufficient enough to print 200 stamps. The sample volume which is delivered in each stamping is around 0.5 to 0.7 ηl. After each printing the pins are sonicated for 2000 milliseconds, followed by washing in sterile water for 3000 milliseconds and drying under vacuum for 2000 milliseconds which is repeated thrice, the final cycle has an extended drying time of 3000 milliseconds. After each 384 well plate the pins are also sonicated thrice for 3000 millise
  conds. During each dip, in order to avoid the possibility of sample sticking to the walls of the pins, which can cause excess sample being deposited on slides will result in run over of samples, the pins are initially blotted on a poly-L-Lysine coated blot pad 10 times with 150 micron spot to spot spacing such that no sample remains along the walls of the pins. The time taken by the robotic gridder to print 7680 samples in duplicate is 36 hours. The cDNAs were printed with a relative humidity between 45 to 50% RH, this is very critical in printing good and reproducible microarrays. And also before printing calibration of wash station, dry station, sonicator and slides across the platen of the slide holder is of utmost importance for uniform spot morphology in all the printed slides. Array printing:Microarraying was by robotic systems we used a microarray robot built by Gene Machines California (genomic solutions). We have used aminosilane coated slides from Telechem these Su
  perAmine, aminosilane coated glass microscope slides have been the most consistent, when printed in 50% DMSO as a printing solution at 45% relative humidity and a constant temperature of 72oF (22oC). We have used quill pens (Arrayit ChipMaker3 microspotting pins from Telechem International Inc.) which were very durable and could reproduce high-quality spots with good precision, all arrays were printed with the same set of ChipMaker 3 pins for more than six months. Following printing, the slides were rehydrated on a steam coming from a 500ml conical flask containing MilliQ water, which was kept boiling on a hot plate. Care was taken such that all the slides were uniformly rehydrated, the slides were just passed over the steam with the array side down 2 to 3 times until a thin layer of mist was formed on the printed side, extreme care was taken not to rehydrate such that the spots run into each other following rehydration the slides were immediately snap dried for 5 sec on a h
  ot glass plate. The slides were allowed to dry and spotted DNA was fixed to slide by UVcrosslinking at 320 mJ using a Stratalinker (Stratagene, Cat# 400071) and by baking at 80o C for 2 hrs. Printed slides were stored in a light-tight box in a bench-top dessicator, with desiccant and were kept at 4o C until they were used for hybridization.
  2. Total RNA was isolated from leaf and panicle samples collected at different time intervals after stress treatment using Trizol (Chirgwin et al., 1979; Chomczynski and Sacchi, 1987) from Life Technologies with minor modifications. The harvested tissues were stored immediately in RNA later (Ambion) and stored at 4oC for 24 hours and after which the RNA latter was removed and the samples were quick frozen in liquid nitrogen and stored at –70oC. Five grams of freshly harvested or frozen tissues were ground in liquid nitrogen to a fine powder, transferred to 50ml tubes with 15ml of Trizol in it. The sample was resuspended well in Trizol and incubated at room temperature for 5 minutes. Cellular debris was removed by centrifugation at 4000 rpm (2700 g) for 15min at 4oC. The supernatant was transferred to a fresh RNAase free 50 ml oakridge tube. To it 3ml of chloroform (0.2 ml/1 ml Trizol) was added and shaken vigorously for 1 minute. After which the suspension was incubated at
  room temperature for 5 minutes. The samples were centrifuged at 4000rpm (2700 g) for 15min at 4oC to remove proteins and high molecular weight DNA, following centrifugation three different layers are observed. The top supernatant layer contains RNA, the intermittent layer is a denatured protein layer, and the lower layer is high molecular weight DNA. The upper aqueous layer was carefully removed without disturbing the protein layer and was transferred to a fresh RNAase free oakridge tube. To this equal volume of isopropanol was added to precipitate the RNA and was incubated at RT for 15 minutes. Following which the precipitated sample was centrifuged at 15,000rpm (21,000g) for 15 minutes to pellet the RNA. The supernatant was discarded and the pellet resuspended in 70% ethanol. The RNA pellet was resuspended in diethylpyrocarbonate (DEPC) treated water. The quality of the total RNA was determined through 1.2% denatured agarose formaldehyde gel and concentration estimated sp
  ectrophotometrically. For labeling  the samples were resuspended in 1/10th volume DEPC treated sodium acetate and 2.5 volumes ethanol. Prior to labeling the precipitated RNA was spun at 12000 RPM for 10 min and washed with 70% ethanol. The RNA was dried in a savant rotary vapor trap and resuspended in RNAase free milliQ water for labeling.
  3. Probes for microarray analysis are prepared from RNA templates by first strand synthesis of cDNA containing amino-allyl-labeled nucleotide dCTP and amino-hexyl modified dATP for both control and stress samples, followed by a covalent coupling to the NHS-ester of the appropriate Cyanine fluor. This method has proven highly efficient for labeling all kinds of tissues from rice for expression analysis.  Superscript III RT (Life Technologies; Cat# L-1014-02) was used to  generates probes with significantly greater activity. Coupling of modified cDNA with fluorescent probes was done by photostable Alexafluor  fluorescent dyes (we have used Alexafluor 488, and 555 for control labeling and Alexafluor 594, and 647 NHS ester dyes for coupling stressed samples), The  photosensitivity of dyes was minimized by avoiding exposure to light during labeling, hybridization, washing, and scanning processes. Upon receipt, these NHS ester dyes were stored in light- proof tubes and stored at -
  20oC until needed. All reactions were carried out in foil-wrapped tubes and all hybridizations and washes in foil-wrapped containers.Labeling procedure First strand amino modified cDNA synthesis recipe step 1:Prior to labeling the Quality of total RNA for both control and stressed experimental sample, were quantified and  40μg of total RNA was resuspended in not more than 10μl DEPC treated water. Anchored oligo DT prim 2.0μl. DEPC treated water was added to make-up a final volume of 30.0 μl.  The above RXN was Incubate for 2 hours at 42o C in a thermocycler.  Alkaline hydrolysis and neutralization step 2:To the reaction from step 1, 15 μl 1N NaoH was added to denature the RNA through hydrolysis from RNA-DNA hybrid and was incubated for 10 minutes at 70o C for complete RNA denaturation. To this 15μl of 1N HCL was added to neutralize the pH and mixed gently. To the above reaction 20μl Sod.Acetate, pH 5.2 was added and mixed gently. Once the  cDNA is generated with amino-m
  odified nucleotides the cDNA was purified to remove unincorporated dNTPs and hydrolyzed RNA, using bind wash elute SNAP columns (Life Technologies) as per manufacturers instructions following which, the cDNA was O/N ethanol precipitated by adding 1/10th volume Sodium Acetate and 2.5 volumes ethanol. First strand cDNA dye coupling step 3:To the aminoallyl and aminohexyl modified cDNA precipitated in step2, was further   centrifuged at 12000 RPM for 10 min to pellet it down and the pellet was washed with 70% ethanol and dried down in speedvac (Savant). The pellet was dissolved in 6μl sterile water. For coupling of the dye, the dye vails (Alexa fluor dyes) were allowed to come down to room temperature in order to avoid condensation of water inside the vial. The dyes were dissolved in 4μl of DMSO. For coupling 10μl of 2x coupling buffer (sodium bicarbonate) was added to modified cDNA to bring the final concentration to 1x, to the above reaction 4μl of the dissolved dye was added and allowed to couple overnight in dark at room temperature. Further, the cDNA was purified by bind wash elute SNAP columns (Life Technologies) as per manufacturers instructions to remove any unreacted free dye molecules.
  4. Hybridization:The dye labeled cDNA once quantified, equal amounts of cDNA from tester and reference samples were pooled and was precipitate by adding 1/10th of Sodium acetate and 2.5 volumes of ethanol, the cDNA populations are spun at  8000 RPM for 10 min, to pellet down the cDNA, extreme care was taken not to disturb the pellet while decanting. The pellet was washed with 70% ethanol and dried in speedvac, and the pellet was directly dissolved in 50μl of low temperature hybridization buffer from sigma. Preparation of slides for Pre-hybridization/ Hybridization:We have developed protocols that give reproducible, high-quality hybridization results while maximizing the measured fluorescence from the array. Aminosilane coated slides bind DNA with high efficiency. Prior to hybridization, the free amine groups on the slide were blocked or inactivated to avoid nonspecific binding of labeled cDNA to the slide which can deplete the probe and produce high background. The slides we
  re blocked in Sodium Borate and Succinic anhydride, a solution of 0.5M Sodium Borate was prepared by dissolving Sodium Borate by heating until it got dissolved and ph was adjusted to 8.0. Simultaneously 900mg succinic anhydride was dissolved in 47ml of 1 methyl 2 pyrillidone (Sigma Aldrich), in a 50ml falcon tube and as soon as the last flake of succinic anhydride was dissolved 2.2ml, 0.5M Sodium Borate was added and was mixed well. Microarray slides stored at 4 deg C were allowed to come to room temperature, the required number of slides were dipped in the above solution and kept for shaking on an orbital shaker for 15 min, and care was taken that the printed side does not touch each other, the slides were placed in opposite direction such that the non printed side touches each other. This was Scaled up if more than 2 slides were processed using a Lipshaw slide holder and jar. After 15 min the slides were washed in 0.1% SDS for 2 min in a clean glass jar (Lipshaw dish stain
  ing assembly) the SDS solution was removed and a brief 10sec wash was performed with milliQ water, and were transferred to a fresh dish jar containing  2XSSC,  the slides were washed twice with 2X SSC for 2 min each. Denaturing and fixing:Denaturation was performed by dipping the slides in boiling hot water at 95o C and incubating them for 2 min. The denatured DNA was fixed by transferring the slides in ice cold 200 pint 95% ethanol (Aldrich), care was taken to avoid shattering of slides due to sudden transfer of slides from boiling hot water to ice cold ethanol. Slides were retained in ice cold ethanol for 2 to 3 min before being transferred to a 50ml falcon tube with a tissue paper at the bottom and the slides. The slides were spun without cap at 1000RPM for 5 min at RT, there is every possibility that the slide coating could peel off but the substrate we selected withstood the pre-hybridization conditions and all the slides processed did not peel. After spinning the slide
  s were immediately used for hybridization. Target hybridization:The labeled cDNA dissolved in low temperature hybridization buffer from sigma, two blocking agents yeast tRNA (Sigma) at a concentration of 20μg per hybridization, and 2μg poly DA (Sigma) per hybridization were used to block non-specific hybridization, these two were mixed with the labeled target and was denatured at 95o C for 5 minutes, during this period the pre-hybridized slides were heated to bring the slides to 45oC for hybridization. After denaturation the labeled probe was applied to a pre-hybridized microarray slide and covered with 22mm x 60mm plastic hybrid-slips (Sigma, Schleicher and Schuell Gmbh). Once the coverslip is properly placed without any air bubble trapped between the slide and the coverslip, the slides were inverted (array side down) and kept inside a 50ml falcon tube, which we used as an makeshift hybridization chamber, a small piece of tissue paper was soaked with 1ml of 3x SSC and was
  kept inside the bottom of falcon tube to maintain humidity during hybridization and to avoid drying of the target, which will make the coverslip stick to the slide and result in failure of hybridization. We placed the 50ml falcon tube in hybridization bottle (Amersham Pharmacia) and with support on two sides with bottle holders these units were dipped in hot water bath set at 50oC. Weight rings were used between the bottles to avoid floating of the hybridization bottles. Hybridization was allowed to carry out for 16 hours in dark at 50oC. Washing:The slides from the water bath were taken out after 16 hours of hybridization and these slides were carefully dipped in wash buffer 1 (1x SSC , 0.03% SDS), the slides were shaken gently until the coverslip come off by itself, in any case care was taken not to take of the coverslips manually, but rather was allowed to soak in the buffer for some more time by shaking gently. Once the coverslips come off, the slides were transferred t
  o wash buffer 2 (0.2x SSC)  and the slides were washed  for 2 min by gently shaking and then transferred to wash buffer 3 (0.05x SSC) these slides were further washed with gently agitation for 5 min. The slides were then transferred to a 50ml falcon tube and with its lid open were spun at 1000 RPM for 5 min in order to dry the slides for scanning. Extreme care was taken that the slides are not dried between transfers.
  5. Scanning of the hybridized slide was done using ScanArray 3000 (Genomic solutions). Hybridized slides are scanned first in the high wave length channel (594nm and 647nm), and then the lower wavelength (488nm and 555nm), as high wave length channel dyes are more susceptible to photo-degradation than lower wavelength channels. Data from each fluorescence channel was collected and stored as a 16-bit TIFF image. To quantify the right intensities slides were scanned as soon as possible after washing. The slides were scanned at appropriate PMT gain and 100% laser power, the slides were scanned at various PMT gains from low to high at 30 microns scan resolution, then appropriate PMT settings for both the dye wavelength was standardized. Scanning was done at PMTs where we could observe 20 to 30 white spots as such the overall intensities are good. This was determined by observing the RI (Ratio Vs Intensity) plot of intensities. Once the right PMT gains for both the channels were
  determined, the slides were scanned at 10micron resolution. During quantification we used the highest threshold limits for spot intensity calculation and ANOVA for performed for these low PMT and High PMT technical replicates. During quantification care was taken that the week spots and saturated spots are flagged off during intensity calculation. To minimize the background fluorescence, care was taken such that the background fluorescence is as low as possible, using appropriate blocking agents. Scanned images generated from microarray experiment were quantified with Quantarray (GSI Lumonics), software
  6. Data from each fluorescence channel was collected and stored as a 16-bit TIFF image. The two images were loaded on to the two different channels of Quantarray and grids were generated taking printing style in to the consideration. Overlay of the two channel intensities revealed the hybridization was good and more or less similar intensities were observed in adjacent in-slide replicates. A total of 48 blocks with 324 features in each were generated with 12 rows and 4 columns in each covering all the ~15550 features including controls. A total of 15552 cells were generated with 5 - 23 pixel spot size adjusted as to cover all the features of the superimposed slide. In house custom perl scripts were written to facilitate the annotation to the features on the array. Annotation including gene function, chromosome Bac/Pac location, map positions, unigene cluster, functional classification, and redundancy were provided to facilitate further analysis with ease. All the grids were
  processed and spots were quantified in the cells eliminating local background. The spots with poor quality in either of the slide were flagged off and were eliminated during the normalization. The intensities captured at the saturated level were excluded from further analysis and quantified spots from each analysis were stored for further analysis.
  7. Rice seeds were surface sterilized, washed and germinated in a growth chamber. Fifteen day old seedlings were transplanted in the field for drought stress induction. The transplanted seedlings were grown in 100% field capacity with 2cm standing water until day 30. Field drought stress was initiated from day 31 at 100%, 60%, 40% and 15% field capacities designated as A1, A2, A3, and A4. It took 8 days for the onset of the actual drought for the experimental plots to reach to their designated field capacities from 100% FC. The irrigation Samples were collected at various stages and drought stress regimes based on the developmental stages of the plant. Rainout shelter was constructed to cover the plots during rainy days to keep the plants from rain during experiment. The rainout shelter was provided with free moving rail tracks to move forward and backward. Proper care was taken to avoid any shade of rainout shelter keep on plants. The drought stress induction was monitored
  through high-precision digital irrigation controllers procured from Netafim Inc (Israel). Irrigation was given taking Plot A1 /AR1 as 100% field capacity, which is a control plot in this experiment, and irrigation was set up in the other plots as shown in the table below with moisture probes/Timer controllers using digital controllers.