Micro Array Practical Method

Practical for Microarray technology Aims: The aim of this practical is to provide an understanding of the modern biotechnology in functional genomics and cancer biology. These approaches are revolutionizing the biomedicine technology, especially in cancer research, influencing diverse areas of biological research from vaccine through to drug discovery process. The practical will provide the opportunity for the students to explore the key technology for modern molecular biologists - Microarray technology, which has been widely used in different biomedical field including cancer research; To be familiar with the techniques used in functional genomics analysis and how large-scale gene functional analysis programmers are designed and implemented in genomic research; To increase the competences required in modern laboratory skills. Objective To study the gene expression profile using Microarray technology. --- Specifically, you (working in pairs) will compare T lymphocytes (a-MF2 for activated MF2 cells) with the same cells but transfected with a gene called Egr-2 (a-Egr2/MF2, activated MF2 cells with a Egr2 gene) to see whether there is any difference of gene expression profile after they are activated. Practical arrangement

Week-1 Practical

Week-2 Practical

Practical topics (13:00-17:00) Lab135

Practical topics (13:00-17:00) Lab135

Monday

Microarray practical-1 – RNA extraction – Measuring RNA concentration

Microarray practical -4 - Array chip pre-hybridization - Array chip hybridization

Tuesday

Microarray practical-2 – Making cDNA and Labeling – RNA on agrose gel

Microarray practical -5 - Array chip washing - Array chip scanning

Wed.

Microarray practical -3 – cDNA purification – Quality control check

Microarray practical -6 -Array analysis: bioinformatics - Instruction for Practical report

Date

Practical Report: You need to hand in your practical report (80%) together with your practical log book (20%) on Wednesday, 13th of December, 2006.

Microarray practical-1: Samples:

- Each pair is provided with one of the following test sample for RNA extraction. Group 1-8: activated MF2 cells (a-MF2) 1

Group 9-16: activated MF2 cells with Egr-2 gene (a-Egr2/MF2) Note: RNA will be degraded if not properly handle it! -Always wear gloves throughout the procedure and avoid making contact with anything (ensure that everything is organised for the experiment beforehand). - Place paper towel on bench working area before starting the experiment -Avoid contact of pipette tips with anything when carrying out the experiment

Procedure-1: RNA extraction with TRI reagent 1.

The cells are already with 500 ul TRI reagent (lysis buffer) on ice

2.

Allow the sample to stand at room temperature for 5 minutes.

3. Add 100 ul of chloroform (200ul per 1 ml of TRI reagent used) to each sample followed by vortexing to mix the sample for 15 seconds. 4. Leave the sample to stand at room temperature for 15 minutes.

5. Centrifuge the mixtures (12000 RPM, 4 Degrees C for 15 minutes). NOTE: It should be expected for each of the samples to form 3 phases upon centrifugation; a red organic phase containing protein (bottom-most layer), an inter-phase containing DNA (middle layer) and a colourless aqueous phase containing RNA (upper-most layer).

6. Transfer the upper-most aqueous phases from sample to fresh eppendorf tubes and then add 250 ul of isopropanol (500ul per 1 ml of TRI reagent used) to the sample.

7. Mix the sample and then allow to stand at room temperature for 10 minutes. 8. Centrifuge the sample (12000 RPM, 4 Degrees C for 10 minutes).----make a mark on the one side of tube, so you know where you expect to see the RNA pellet! because following centrifugation, the RNA precipitate will form a pellet on the side and bottom of the tube for the sample.

9. After centrifuging, remove the supernatant and then the RNA pellet should be washed by adding 500 ul of 75% ethanol.

10. Mix the sample thoroughly by vortexing and then centrifuge (7500 RPM, 4 Degrees C for 5 minutes). 11. Remove supernatant from sample as much as possible (otherwise it will not be dissolved in the next step!)

2

12. Air-dried for 10 minutes. NOTE: When drying the RNA pellets, care should be taken to ensure that the pellets do not dry completely, as this would decrease the solubility of the pellets !

13. Finally, add 100 ul of DEPC (Di-Ethyl-Pyro-Carbonate) treated water to sample pellet. To facilitate dissolution, mix sample by repeated pipetting for 5 minutes.

Note: You need to take 5ul from the RNA sample for running agarose gel (at procedure-4, Practical -2 tomorrow), label the tube with your group number and give it to Helen.

Procedure-2: Measuring RNA with spectrophotometer -Use a spectrophotometer in order to determine the quality of the extracted RNA samples. It is noteworthy that the quality of RNA is one of several important determinants, governing the success of micro-array experiments. - The criteria for determining the purity of RNA is based on the 260/280 ratio which is a measure of the extent to which the RNA sample is free from DNA and protein. A 260/280 ratio of > 1.7 indicates that the RNA is of acceptable purity/quality. - Procedure to measure the purity of each of the extracted RNA samples, 1) 990 ul of water add in a corvette. 2) 10 ul taken from your RNA sample and add to the same corvette, mix well. 3) The cuvette will be then placed in the complementary slot on the spectrophotometer and record the following from the spectrophotometer: a) Record the reading for the ration: 260/280. b) Record the reading for the RNA concentration in the corvette (ng /ul) 4) Calculate the actual concentration of your RNA sample (ug /ul). - The following table shows the results of the calculations as some examples with dilution factor: 100 (10 ul in 1000 ul of water: 1000/10=100) Reading (examples)

Concentration ( ug/ ul)

260/280 Ratio (examples)

3

5.6 ng/ul

11.2 X 100 (dilution factor) / 1000 =

2.17

19.7 ng/ul

1.12 6.8 X 100(dilution factor) / 1000=

1.96

0.68

Note: 1) Y need to have total RNA for one sample about 50 ug for the experiment, so if the total RNA (total ul x concentration ug/ul) is < 50 ug, then you need to get relevant RNA from Hellen / Dr. Li.

2) You need to dry down the sample if the RNA concentration is < 0.5 ug/ ul, because you will need about 40-50 ug in a volume less than 8 ul, which will be required for the Practical-2 (tomorrow) .

Procedure-3: To get more concentrated RNA (only if it is needed!) 1) Take about 40 ug (40-50ug) ug of your RNA in one eppendorf tube-1 (how much volume you need? e.g if your sample contraction 1.12 ug/ul, then you will need the volume: 40 /1,12= about 40ul), 2) Take another eppendorf tube-2 with water as control to measure the volume for the evaporation, which should be the same volume as tube-1, 3) Label with your group number 4) Put both tubes (water tube and sample tube) with opened lid in a rotator evaporator (located at Lab 128), 5) make a mark on the one side of tube, so you know where you expect to see the RNA sample!

6) The rate for the evaporation is about 1ul/min. About every10 min, check the volume for the water tube (tube-2) until the volume is 8 ul or less.

4

Microarray practical-2

Procedure- 4: Cyscribe First-strand cDNA Labelling

- You should have two tubes: one containing control RNA sample: (MF2 RNA) which is on the ice already and the other containing test RNA sample (you extracted) for this experiment. Both need the volume
- THIS MUST BE DONE FOR EACH OF THE 2 TUBES!!!! A) Primer annealing for each of two RNA: 1.

8 ul RNA (add water if less than 8ul) + 3 ul Oligo-DT ---------------------------11 ul in total

2. Mix gently by pipetting up and down. 3. Incubate reactions at 70 degrees C for 5 minutes (use heating block). 4. Cool reactions at room temperature for 10 minutes to allow the primer and the mRNA template to anneal. 5. Spin down reactions for 15 seconds in a micro-centrifuge. B) Extension Reaction: 1) Add the total 9ul of “cy-3 mix” to the 11 ul of control RNA(MF2 RNA) 2) Add the total 9ul of “cy-5 mix” to the your extracted 11 ul of test RNA Each mix contain: 5 X Cyscribe Buffer 0.1 M DTT dCTP Nucleotide Mix dCTP Cye-Dye-Labelled Nucleotide Cyscribe RT (enzyme)

4 ul 2 ul 1 ul 1 ul 1 ul

The only difference for the mix are: Cy-3-dCTP used for the control RNA, Cy-5-dCTP for the test RNA. Total = 9 ul (above reagents) + 11 ul (RNA + Oligo-DT) = 20 ul. 3. Mix by gentle pipette, then centrifuge for 30 seconds in micro-centrifuge. 4. Incubate the reactions at 42 degrees C for 1.5 hours. (after incubation, place reactions on ice for next step) 5

Note: during the incubation, you need to do procedure-5

Procedure- 5: RNA quality check by argrose gel- demonstration - You will see the demonstration how to run the agrose gel when you are doing procedure-3B. - In the end of the experiment today, you will get the picture for your RNA taken from the agrose gel and you need to analysis the RNA quality to see how much degradation of the RNA.

Procedure-6: Degradation of unlabelled RNA (continue from procedure-4): 1. Adjust water bath to 37 degrees C. 2. Add 2 ul of 2.5 M NaOH to each cDNA reaction from above procedure-3B. 3. Mix by vortexing and spin for 15 seconds in a micro-centrifuge. 4. Incubate reactions at 37 degrees C for 15 minutes 5. Add 10 ul of 2 M HEPES-free acid to the reactions 6. Mix by vortexing and spin for 15 seconds in micro-centrifuge 7. The reactions are now ready for purification (practical -3, tomorrow), give to Helen to store in -20.

6

Microarray practical -3 Procedure- 7: Purification of Labelled cDNA: 1. Add 500 ul of capture buffer to 1 GFX column 2. Add both cDNA samples (already with Cye-5 and Cye-3 in them) into the column and mix 3. Centrifuge the column – 13,000 rpm – 30 seconds 4. Add 500 ul of wash buffer to the column – centrifuge 13,000 rpm, 30 seconds 5. Discard liquid in collection tube 6. Add 500 ul of wash buffer to the column – centrifuge at 13,000 rpm, 30 seconds 7. Discard liquid 8. Place column back into the collection tube + centrifuge at 13,000 rpm for 10 seconds 9. Transfer column to a fresh eppendorf tube + add 30 ul of elution buffer to the tip of the column 10. Incubate at room temperature for 5 minutes 11. Centrifuge at 13,000 rpm for 1 minute 12. You will get about 30ul of elutes after centrifugation. Take 1ul of elutes for labeling efficiency check at procedure-8. 12. Dry down the cDNA using a rotator evaporator until less than 1 ul. (the procedure is the same as in procedure-3, make a mark on the one side of tube, so you know where you expect to see the pellet!!!

Procedure- 8: Labeling efficiency check – – –

You will be provided one microscope slide Put 0.5ul of labeled cDNA on the slide Add 10ul of water into the rest of the tube (about 1/20 dilution). And put 1ul from this diluted cDNA on the slide. – Dry the slide, label it with the group number, and give it to Helen/Moshin, and we will do the scan to see how the labeling efficiency is.

7

Microarray practical -4: Note: 1) Each group will be provided an array chip which contains about 15,000 genes/ or 283 genes. Each gene was arranged in a specific position on the glass slide, and you will use this chip to hybridizated with your cDNA 2) Avoid to touch the surface of the chip which contains the DNA genes.

Procedure- 9: Pre-hybridization of chip (to get rid of background) 1. Set water bath at 42 degrees C 2. label the chip with test sample information (eg. a-MF2 or a-EGR) and group number, and the date. 3. Add 500 ul of pre-hybridization buffer to middle of the chip (be cautious of air bubbles) 4. Place the cover-slip carefully on the chip. Place cover-slip horizontally on chip so that hybridization buffer covers the chip and air bubbles are expelled out 5. Give the chip to Helen/Moshin, and the chip will be put into cassette and incubate at 42 degrees C for 1-2 hours until you prepare the stuff for hybridization step see below.

Procedure- 10: Hybridization of the chip (cDNA hybridizated with DNA genes): 1. Re-suspend cDNA (find the pellet first!) with “blocking mix” which contains 6 ul of Cot DNA+1.5 ul of oligo-dA 2. Denature at 95 degrees for 2 minutes (use heat block) 3. briefly centrifuge 10 seconds 4. incubate at 75 degrees C for 45 minutes (use heat block) 5. Add “hyb-mix”(which contains 7.5 ul of hybridization buffer + 15 ul of 100% formamide) to the above tube. Mix well + spin for 15 seconds 6. Take off cover-slip (do not touch chip) from the pre-hybdizated chip (get from Helen/Moshin) 7. Add whole sample from step-5 to the middle of the chip. Place cover-slip carefully on the chip, then give to Helen/Moshin 8. Incubate the chip in a chamber , overnight in 42 degrees C water bath. Note: Please bring the memory stick /or one CD tomorrow, so we can save the image for you when we scan the chip for your results.

Microarray practical -5 8

Procedure- 11: Washing (to get rid of unbound cDNA) and Scanning 1. Add about 300-400ml of water into the wash container, 2. Place chip in the wash container slot, let cover-slip go away it, and 3. Use the R100 – Luckham shaker to perform the following washes Buffer (300-400ml) 1 X SSC + 0.2% SDS 1.1 X SSC + 0.1% SDS 0.1 X SSC

Time 5 minutes 5 minutes 5 minutes

No of times X1 X1 X1

1. After final wash, empty wash container + replace with water. Place the chip into it 2. Place chip in 50 ml tube, put it in the centrifuge, make sure it has been balanced ! 3. centrifuge 1000 rpm for 20 seconds, Cover tube with aluminium foil 7. The chip is now ready for scanning. Note: 1) Please bring the memory stick /or one CD with you, so we can save the image for your results! 2) We will do the scanning in the Microarray Lab in Cancer Institute.

9

Microarray practical -6 Procedure- 12: Array analysis- Using GoMiner to categorise Genes according to their biological functions Purpose of Software: Typical microarray experiments generate vast amounts of data, making it difficult to interpret for the biologist. If the biologist wishes to extract biological meaning from such overwhelming data, the data itself must be further mined. Go-Miner is one of many tools used to “make sense of the data”. More specifically, it categorises genes from any given data set into their respective function at the biological, cellular and molecular levels. Accessing GoMiner: To access GoMiner, take the following steps: 1. Go to “My Computer” 2. Double click the drive labelled as “L:drive depapps on academic Windsor” 3. Next, double click the “BL” folder 4. Then double click the “GoMiner” folder 5. Opening the “GoMiner” folder will display the files for gominer. Once opened, double click the “GoMiner.jar” file. 6. This should open up the software and the interface should be visible Using GoMiner: When Gominer is opened, the user will be prompted with the following options: Database: Organism: Data source: Evidence Codes: Click on the “Organism” option and select “mus-musculus”. NOTE: Leave all 3 remaining options as they are Next, click on the following tick boxes: Lookup settings Enhanced names (Uniprot only) Synonym Cross reference To load the gene-file of interest, click on the “total” option. This will give the user the option to browse their file of interest. This is the point when you must input the gene file (for the purpose of this practical, use the “down reg T2 genes for gominer.txt” file or the “up reg T2 for gominer.txt” file – both files are located in the “GoMiner” folder in the L drive)

10

Once the input file has been browsed, select the “same as total” tick box next to the “changed” option. Next click the “process” button NOTE: Make sure to wait until it has fully processed. Also, after it has processed, another progress bar will appear. Make sure that this progress bar is also fully complete so do not click “exit”. The ontology should now appear on the right side of the interface and on the left side, the genes should be listed. The ontology should look like this: + Biological process +Cellular component +Molecular function Exporting GoMiner Results: To export the results, click on “biological process” on the ontology, and then right click on the mouse and select the option to “export genes by category”. The user will be prompted to enter a name of the file to save the data in. Enter any name for the file but make sure that it ends with the .txt extension. The user can now view the results. To import the results into excel, copy the contents of the text file and paste it into excel.

11

Instruction for practical report The main sections of the report should be as follows: 1. Title 2. Abstract 3. Introduction 4. Material and Method 5. Results - You need to include at least four results including the pictures/or table produced during the experiment: a) RNA quality and quantity b) cDNA labelling efficiency check c) Array chip hybridization results d) Present the results produced from the Gominer software analysis. 6. Discussion –

You need to discuss your result produced from the practical

–

Also select any 5-10 genes from by Gominer analysis, which are relevant to your experiment system, and search on the internet for the information to explain why they are highly expressed or lower expressed or no changes.

1. References 2. Appendices

Note:

Hand in your practical report (worth 80%) together with your practical log book (worth 20%) on Wednesday, 13th of December, 2006.

12