A: Background Information
For many years many people have been wondering which genes have cells that cause cancer, which don't, and how would we know? The answer is Microarrays. Microarrays play a very important role in the world of science today. Microarrays are a new research tool that enables scientist to see how genes, tissues, cells, are used by taking gene expression that get transcribed into RNA which gets translated into a protein. The major steps which are used to prepare a microarray experiment for tissues are to first isolate the DNA, then amplify the genes. After that a robot will spot individual gene sequences onto a DNA chip. Then one must obtain mRNA tissue from one tissue of interest and choose to label it with the color green, and another tissue of interest and label it red. Colors play a vital role in these experiments because it is how scientists will figure out which tissues, or any other sample, are being used. These mRNAs will be applied to the chips and the results will be analyzed to find out which tissues have been used in the genes. Scientist can finally figure this out by looking at the colors, mainly green, red, yellow, and black. Green or red will be the color of the spot depending on which tissue or cell is chosen for each. Yellow will be the color of the spot if it is transcribed in both and black will be the color of the spot if it is transcribed in neither. Unfortunately, most schools do not have enough money to spend on real microarrays for this lab, so in this lab it will be a simple version, however it will work just as well and give us the results needed.
B: Objective & Purpose
The purpose of this lab is to analyze the slide with different samples of cancerous and noncancerous genes to find out which are tested positive for lung cancer and which are not. This lab is very important to the world of science as well because many different scientist and companies use this same lab to do either tests. However, they're labs will differ slightly since they are using expensive Microarrays which also use different colors such as green, yellow, and red. Nonetheless, tests like these can help find cures of different diseases, cancers and many other sicknesses.
C: Procedures
This lab will be done is pairs. First, one must have a microarray slide and make sure that they do not contaminate it by touching the top with anything, hold it by the sides. After placing the microarray on the lab table, obtain the DNA tubes from the 70 degree water bath. Using a micropipet of 15 ul, take 15 ul of each solution and place it in the corresponding circle on the slide, in addition to using a new tip for each spot. The next step is to hybridize the microarray with the cDNAs normal lung tissue and cancerous lung tissue. The bottle which is given to share between both groups contains a solution labeled cDNA from lung cancer cells and normal cells mixed together. When dropped into each spot, it will be a blue dye if it is active in the normal cells and a pink dye if it is active in the cancer cells, in addition to purple when active in both. First, one must be care that is solution does not get into eyes, skin, or clothing because it will be an irritant. Carefully add 20 ul of the "Hybridization Solution" to all the spots, and do not allow the tip of the pippet to touch the spots so you can reuse it for the rest. After waiting about a minute, one will be able to see and note their results (the colors). Once done recording the results, one lab partner will return the tubes to the 70 degree water bath while the other lab partner can rise off the results with water and use a paper towel to clean off the slide and bring it to the front.
D: Controls, Variables & Prediction
In this lab there will be six genes to study while using a "microarray". Gene 1 C4BPA - the protein this gene codes for helps initiate part of our immune system; Gene 2 OCD1 - the protein this gene codes for is an enzyme in the pathway that catalyzes the conversion of ornithine to putrescine; Gene 3 FGG - the protein this gene codes for is a part of fibrinogen and is found in blood; Gene 4 HBG1 - the protein this gene codes for is normally expressed in the fetal liver, spleen and bone marrow; Gene 5 SIAT9 - the protein this gene codes for catalyzes the formation of another protein, GM3, which plays a role in cell differentiation, controlling cell growth, and maintaining the shape of cells; and finally, Gene 6 CYP24 - the protien this gene codes for enzymes that catalyze many reactions involved in drug metabolism and the making of cholesterol, steroids, and other lipids. In addition to these genes, one will also add the hybridization solution to get your results.
Observations:
Discussion:
A: Analyze and Explain Data
The results which my lab partner and I extracted from the lab were transcribed from DNA to RNA to cDNA and are as followed (and in the picture above); #1 Gene (Pink) was expressed in lung cancer cell, #2 Gene (Purple) was expressed in both cell types, #3 Gene (Blue) was expressed in the normal lung cell, #4 Gene (Clear) was not expressed in any of the cells being a non-protein gene, #5 Gene (Pink) was expressed in lung cancer cell, and #6 Gene (Blue) was expressed in the normal lung cell.
B: Possible Sources of Error
Some possible sources of error which could happen in this lab are if you do not have a steady enough hand to put the drops onto the different spots or if the measurements for the substances are not correct with the micropippets. In addition, the slides which the drops of genes are on is contaminated by other DNA or if the tip of the pippet is contaminated by touching other genes/not changing after touching different genes. Both of these errors maybe small however they cause a huge problem in the results which would be produced from the lab.
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