QUANTITATE LEVEL OF TRANSCRIPT OF THREE HEAT SHOCK GENES IN ACROPORA MAURITIANA USING CDNA MICROARRAY

TECHNIQUES
MOODELLY Sheyne - 1011412

ASKOORUM Chaveena - 1014340

KOWNDEN Vanitha - 1000023 JUHOOR Khalid Khan - 1011982

Aim
Quantitate level of transcript of three heat shock associated genes- hs1,hs2,hs3 in Acropora mauritiana at 23 and 26 oC using cDNA microarray techniques

Specific objectives
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Collect Acropora Mauritiana which is in good health condition Acclimatize in circulated shaded tank having filtered seawater for four days. Exposed the coral to different temperature 23,26 oC Blast coral tissue and obtain mRNA by centifugation Used Microarray screening to quantitate the level of transcript of the three heat shock genes

BACKGROUND OF CDNA MICROARRAY

cDNA microarrays developed in the Brown and Botstein labs at Stanford and this technologies have been developed to exploit DNA sequence data and yield information about gene expression levels Studied different aspects of gene expression such as expression at the transcription or translation level, and subcellular localization of gene products. To date, attention has focused primarily on expression at the transcription stage, i.e., on mRNA or transcript levels.

Microarrays make use of complementary base-pairing property of DNA molecules, and hybridization is used which involved the annealing of nucleic acid strands from different sources according to the base-pairing rules

cDNA microarrays consist of thousands of individual DNA sequences printed in a high-density array on a glass microscope slide using a robotic arrayed

METHODOLOGY
1. Acclimation and tissue removal

Collect colonies of approximately 2 cm2 X 2 cm2 to a depth of approximately 3 m at low tide which is exposed to minimum disturbances The colonies were transferred to different tanks containing filtered sea water which was well aerated and exposed to 2 different temperature (23 and 26 oC) for 24 hr which were adjusted using a thermostat Coral tissue is blasted using small, forceful, intermittent jets of refrigerated distilled water delivered rapidly by a Water Pik. The tissue is removed within large polyethylene bags to prevent loss by splattering then poured into a beaker; the bag is rinsed twice with distilled water and added to the beaker

METHODOLOGY
2. Fabrication of cDNA microarray

Used Reverse transcriptase-PCR where Quanti Tect Reverse Transcription Kit was used for the reverse transcription process which is a procedure taking only 20 minutes and comprises 2 main steps: Elimination of genomic DNA : The purified RNA sample is briefly incubated in gDNA Wipeout Buffer at 42C for 2 minutes to effectively remove contaminating genomic DNA Reverse transcription : The entire reaction takes place at 42C and is then inactivated at 95C. Quantiscript Reverse Transcriptase having high affinity for RNA This high RNA affinity, in combination with Quantiscript RT Buffer, enables high cDNA yields. RT Primer Mix ensures cDNA synthesis from all regions of RNA transcripts, even from 5' regions.

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METHODOLOGY In general, reverse transcriptase is a multifunctional enzyme with 3 distinct enzymatic activities: RNA-dependent DNA polymerase, a hybrid-dependent exoribonuclease (RNase H), DNA-dependent DNA polymerase.

METHODOLOGY
Performed PCR to identified gene

The cDNA sample was used as template with its specific primer to produces microarray containing the immobilized genes The specific gene sequence of the three gene HS1,HS2 and HS3 was obtain from gene bank and specific primer was developed from Primer3 software

2.0 l of the cDNA template, 1.5 mM of forward primer, 1.5 mM of reverse primer, 5 l of nucleases free sterile water were used in the PCR reactions
Check PCR products by agarose gel electrophoresis Analyze 2 l of PCR product on a 2% agarose gel. Gel imaging allows a rough quantitation of product while giving an excellent characterization of the product. Band size and number was observed in the PCR products will enable us to know if the specific cDNA is present

METHODOLOGY
Purification of PCR products Fill 96 well V-bottom plates with 200ul per well of ethanol/acetate mix. Transfer 100ul per well of PCR product into V-bottom plates and mix by pipetting a volume of 75 l per well four times Place the plates in -80C freezer for one hour or store overnight at 20C. Then melt any ice, which may have formed in the wells. Load the plates into a centrifuge with a horizontal microtiter plate rotor and spin at 2600 x g for 40 minutes at 4C. Aspirate the supernatant from each well using the Immunowash plate washer Allow the plates to dry overnight in a closed drawer Resuspend the PCR products Add 40 l of 3X SSC per well. Seal plates with a foil sealer Place the plates in heat sealable bags with paper towels moistened with 3X SSC Place the bags in a 65C incubator for 2 hours

METHODOLOGY
Side coating Slides coated with poly-L-lysine have a surface that is both hydrophobic and positively charged. The hydrophobic character of the surface minimizes spreading of the printed spots, and the charge appears to help position the DNA on the surface in a way that makes cross-linking more efficient. Printing Pre-clean the print pens according to the manufacturer's specification. Load the printer slide deck with poly-L-lysine coated slides Thaw the plates containing the purified PCR products and centrifuge briefly, two minutes, at 1000 rpm in a horizontal microtiter plate rotor to remove condensation and droplets from the seals before opening. Transfer 5 to 10 l of the purified EST PCR products to a plate that will serve as the source of solution for the printer.

RNA EXTRACTION

Tissue Harvest The tissue must be immobilized as quickly as possible after sampling to prevent RNA expression changes due to technical stress and to prevent degradation by Rnases. Snap Freezing in Liquid Nitrogen:

1) Following immersion, keep the tissue in the Nitrogen until the procedure is completed.
2) Upon completion of the harvest procedure, transfer the tissues to empty falcon tubes stored on dry ice. 3) Keep the tissue frozen until the homogenization procedure is ready to be performed.

Immersion in RNA Later: 1) Upon extraction, immediately slice the tissue into pieces no wider than 0.5cm (sample must be small so solution can penetrate) and drop into RNA Later. (The volume of RNA Later should be at least ten times the volume of tissue)

2) Store the tissue (until homogenization) according to the following: Initially overnight at 2-8 C, Then indefinitely < -20 C, up to four weeks at 2-8 C, up to 7 days at 2-8 C, up to 1 day at 37 C.
* For cultures of cells, pellet out of growth media, wash 3 X PBS, and resuspend in RNA Later. (Do Not Freeze!)

Homogenization
1) For tissues that are snap frozen or slightly in excess, the homogenization of the tissue should be done by mortar and pestle (cooled to temp in a liquid nitrogen bath).

2) At the same time, transfer at least 1mL TRIZOL / 100mg tissue to be homogenized into a falcon tube 3) Transfer the tissue to the pestle and grind until a layer of very fine dust is all that is left.
4) Use an RNase free spatula to transfer the dust to the TRIZOL solution. Be sure to get as much dust as possible. 5) Vortex mixture thoroughly. * For cultures of cells (suspended in solution), quantify, pellet the cells, and resuspend in TRIZOL at a volume of 5 x 10^6 cells / 1mL TRIZOL. 6) Once homogenized, aliquot the solution to eppendorf tubes. 7) Leave in TRIZOL at room temp for five minutes, and then continue on with phase separation.

Phase Separation 1) Add 200ul chloroform / 1mL TRIZOL (originally used), vortex for 15 seconds, and leave at room temp for 2-3 minutes. 2) Centrifuge samples at 12,000g for 15 minutes at 2-8 C. RNA Wash and Re-suspension 1) Following centrifugation, remove the supernatant. 2) Wash RNA pellet with 80% EtOH / 1ml TRIZOL (originally used) and vortex. 3) Centrifuge samples at 7,500g for 5 minutes at 2-8 C. 4) Remove supernatant. Allow remaining EtOH to air dry for 2-3 minutes. 5) Transfer tubes to 70 C heat block and let sit for 2-3 minutes. 6) Dissolve the pellet in 81ul of DEPC water- DEPC-treated (and therefore RNase-free) water is used in handling of RNA in the laboratory, to reduce the risk of RNA being degraded by Rnases. DNase Treatment 1) Add 8ul of 10X DNase I Buffer. 2) Add 2ul of DNase I Enzyme. 3) Vortex, quickly spin and incubate at 42 C for 25 minutes.

RNeasy Column Purification (Using Qiagens RNeasy Protocol) Qiagen RNeasy columns have proven to be an extremely reliable method for purification of microarray quality RNA. Special kits are available for processing fibrous or lipid tissues. DNAse treatment is easily performed on the column to eliminate genomic DNA contamination.
1) Add 350ul Buffer RLT (with BME-10ul/ml Buffer RLT). 2) Add 250ul 100% EtOH. 3) Apply entire volume to RNeasy column and spin full speed for 1 minute. 4) Reapply entire volume to RNeasy column and spin full speed for 1 minute. 5) Transfer column to new 2ml collection tube. 6) Add 750ul Buffer RPE and spin full speed for 1 minute. 7) Discard flow-through and add 750ul Buffer RPE and spin full speed for 1 minute. 8) Discard flow-through and spin full speed for 1 minute. 9) Transfer column to new labeled 1.5ml Eppendorf tube. 10) Add 56ul DEPC H2O and let sit for 2 minutes. 11) Spin at full speed for 2 minutes. 12) Discard column and transfer tube to ice.

Quantification and Quality Control 1) Quantify each sample using spectrophotometer.

The target mRNA is labeled using reverse transcriptase. During reverse transcription reaction, fluorescent cDNA was generated by incorporation of either Cy3-dCTP (green) or Cy5-dCTP (red) on the sample mRNA with oligo (dT) primer. The primer is annealed to the RNA. A 0.2 ml thin wall PCR tube is used which will be used latter in the PCR cycler. The reaction is done in a 17 l mixture as follows:

Component Total RNA Addition for Cy5 labeling 150-200 g Addition for Cy3 labeling

50-80 g
1 l To 17 l

Oligo dT primer (2 DEPC H2O

g/ l)

1 l To 17 l

Heat (at 65C) for 10 minutes and cool on ice for 2 minutes. Then add of the mixture either Cy5-dUTP or Cy3-dUTP nucleotides. The mixture is well mix using a pipette and a brief centrifuge spin is used to concentrate it at the bottom of the tube. Incubate for about 30 minutes after which superscript II is added and is further incubated at 420 C for 30 to 60 minutes.
Note that Superscript polymerase is very sensitive to denaturation at air/liquid interfaces, so special care should be taken to suppress foaming when handling this reaction. Ethylenediaminetetraacetic acid (EDTA) is added followed by NaOH. Next Incubation is done (60 minutes) to hydrolyze residual RNA and then is cooled to rtp.

Ethylenediaminetetraacetic acid (EDTA) is added followed by NaOH. Next Incubation is done (60 minutes) to hydrolyze residual RNA and then is cooled to rtp. Note that the NaOH should be pure, as slight contamination can result in a solution that can degrade the Cy5 dye molecule thus turning the solution yellow. HCl is added to neutralize the mixture. The labeled cDNA is then desalted. The mixture is mixed and then is spun for 10 minutes.

For recovery, the concentrator is inverted over a clean collection tube and is spin for 3 min. In the presence of contaminants; the cy5 labeled cDNA will form a gelatinous blue precipitate that is recovered in the concentrated volume. A fraction of the Cy5 labeled cDNA is taken for analysis.

The probe is then run on a 2% agarose gel with dimension in Tris Acetate Electrophoresis Buffer (TAE).
After electrophoresis, the gel is scanned on a Molecular Dynamics Storm fluorescent scanner.

HYBRIDIZATION
Hybridization preparation For each slide, combine Cy3- and Cy5- labeled cDNA into one tube and dry them down in a Speed- Vac or other centrifugal vacuum concentrator. For each slide, clean a cover slip by spraying it with 70% ethanol. Resuspend the dried, labeled cDNAs in 6 l of RNase-Free water. Add Amersham Biosciences microarray hybridization solution, 100% formamide, and PolyA. Denature the labeled cDNAs at 94 C for 2 min and spin for 30 sec.

Hybridize fluorescent cDNA to slide Pipette 30 l of the labeled-cDNA solution on an area of the slide that does not contain any spots. Place the clean cover slip on the slide. Make sure bubbles are not trapped under the cover slip and that all of the arrayed area of the slide is covered with hybridization buffer. Place the slide in a humid hybridization chamber. Seal the chamber with parafilm. Incubate the slide for 1218 h in a 42 C water bath. Protect the chamber from light.

Post-hybridization washes Preheat a solution of 2x SSC (saline-sodium citrate), 0.1% SDS (sodium dodecyl sulphate) (Buffer 1) and a solution of 1x SSC, 0.1% SDS (Buffer 2). Transfer the slide to a cradle in a staining jar containing Buffer 1. Wash the slides in Buffer 1 and then Buffer 2 for 5 min each with gentle rocking at room temperature. Cover the staining jar with aluminum foil to prevent photo bleaching of the dyes. Fill three staining jars with 0.1 SSC at room temperature and dip the cradle into these three jars for 5 sec each. Do not allow the slides to dry between washes. Use a low-speed centrifuge that accommodates microarray slides to spin-dry the slides. Place a dry paper towel on the plate holder to absorb the excess liquid, and then place the cradle with washed slides on the plate holder. Balance the rotor and spin for 1 min.

VISUALIZATION

Input: Laser image scans (data) and underlying experiment hypotheses or experiment designs (prior knowledge) and converts multiple colors to values of just 2 colors. Output: Conclusions about statistical behaviour of measurements and thus the test of the hypotheses or knowledge. The results are derived automatically from data (machine learning perspective) for subsequent model fitting

The intensity of the signal is estimated using a calibrated color scale and exact intensityof the signal is determined for each gene.

REFERENCES

http://www.columbia.edu/~bo8/undergraduate_research/p rojects/sahil_mehta_project/work.htm# http://www.genome.gov/10000533

http://www.ncbi.nlm.nih.gov/About/primer/microarrays.htm l

METHOD FOR DETERMINATION OF CORAL TISSUE BIOMASS AND COMPOSITION http://www.aslo.org/lo/toc/vol_15/issue_5/0822.pdf