Professional Documents
Culture Documents
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
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)
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).
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 !
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.
- 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)
3
5.6 ng/ul 11.2 X 100 (dilution factor) / 1000 = 2.17
1.12
19.7 ng/ul 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)
4
Microarray practical-2
- 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 </ or = 8ul of RNA/sample.
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
The only difference for the mix are: Cy-3-dCTP used for the control RNA,
Cy-5-dCTP for the test RNA.
5
Note: during the incubation, you need to do procedure-5
- 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.
6
Microarray practical -3
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- 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)
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
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
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:
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
To import the results into excel, copy the contents of the text file and paste it into
excel.
11
Instruction for practical report
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