Qubi/biol202 - Revision history

imported>Dphilli at 02:49, 7 August 2013

2013-08-07T02:49:13Z

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	*Guessing is not the best way to describe gene clusters. Statistics has a variety of ways to measure correlation. We will use Pearson's Correlation Coefficient, '''''r'''''.		*Guessing is not the best way to describe gene clusters. Statistics has a variety of ways to measure correlation. We will use Pearson's Correlation Coefficient, '''''r'''''.

	*Excel easily measures correlation and Pearson's r. Like most calculations in Excel, you simply click on an empty cell, type "=", write a formula, and indicate what range of cells you wish to perform the calculation on.		*Excel easily measures correlation and Pearson's '''''r'''''. Like most calculations in Excel, you simply click on an empty cell, type "=", write a formula, and indicate what range of cells you wish to perform the calculation on.

	[[File:Bio_202_8.jpg\|thumb\|framed\|100px\|center]]		[[File:Bio_202_8.jpg\|thumb\|framed\|100px\|center]]

imported>Dphilli at 02:48, 7 August 2013

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	'''Question L:''' Much of the previous questions in this lab were just preliminary work leading up to one		'''Question L:''' Much of the previous questions in this lab were just preliminary work leading up to one
	big question: ''Which genes form interactive networks?'' Let us now attempt to answer that! '''Table 5'''		big question: ''Which genes form interactive networks?'' Let us now attempt to answer that! '''Table 5'''
	showed the behavior of yeast genes under glucose deprivation. The correlation data (Pearson's correlation, '''''r''''') can are interpreted in '''Table 7''' as distances. The Table 7 data shows large values for disparate genes and approaches 0 for the ideal case where two genes are expressed in exactly the same levels in varying conditions. This then can be graphically shown as a dendrogram (tree) where similar expression gives close tree-distance. Three genes: NUP 145, COX4, and MLP1 still need to be placed on the dendrogram. Using the pairwise distances from '''Table 7''', place these three genes in the proper location within the tree.		showed the behavior of yeast genes under glucose deprivation. The correlation data (Pearson's correlation, '''''r''''') can are interpreted in '''Table 7''' as distances. The '''Table 7''' data shows large values for disparate genes and approaches 0 for the ideal case where two genes are expressed in exactly the same levels in varying conditions. This then can be graphically shown as a dendrogram (tree) where similar expression gives close tree-distance. Three genes: NUP 145, COX4, and MLP1 still need to be placed on the dendrogram. Using the pairwise distances from '''Table 7''', place these three genes in the proper location within the tree.

imported>Dphilli at 02:40, 7 August 2013

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	==Introduction==		==Introduction==
	[[File:Bio_202_fig_1.jpg\|thumb\|frameless\|'''Figure 1.''' <sub>The first stage of microarray analysis is growth of controlgroup cDNA containing one color dye and growth of experimental group cDNA containing another color dye. The mRNA from a population of cells on the left (represented by a single budding cell) is converted to red cDNAs. In parallel, a population of cells on the right has its transcriptome converted to green cDNAs.</sub>]]		[[File:Bio_202_fig_1.jpg\|thumb\|frameless\|'''Figure 1''' <sub>The first stage of microarray analysis is growth of controlgroup cDNA containing one color dye and growth of experimental group cDNA containing another color dye. The mRNA from a population of cells on the left (represented by a single budding cell) is converted to red cDNAs. In parallel, a population of cells on the right has its transcriptome converted to green cDNAs.</sub>]]
	'''Gene expression''' is the transcription of a DNA template into RNA molecules. In a multicellular organism, the subset of genes that are expressed defines and gives rise to a specific tissue or cell type. In a single-celled organism (such as '''Figure 1''', ''Saccharomyces cerevisiae'', the baker’s yeast), genes are turned on and off depending on environmental conditions. In this laboratory exercise, we will use bioinformatics techniques to identity genes that are expressed in the yeast during glucose starvation.		'''Gene expression''' is the transcription of a DNA template into RNA molecules. In a multicellular organism, the subset of genes that are expressed defines and gives rise to a specific tissue or cell type. In a single-celled organism (such as '''Figure 1''', ''Saccharomyces cerevisiae'', the baker’s yeast), genes are turned on and off depending on environmental conditions. In this laboratory exercise, we will use bioinformatics techniques to identity genes that are expressed in the yeast during glucose starvation.

	Traditionally, gene expressions are studied one gene at a time using blotting techniques. For example, in a '''Northern Blot''' experiment ('''''Figure 4a'''''), the whole messenger RNA (mRNA) content of a cell is extracted and loaded on a solid gel slab. Different mRNA molecules are then separated using electrophoresis and transferred to a nitrocellulose sheet. To identify if a gene is expressed, a radioactively (or fluorescently) labeled oligonucleotide probe that is specific to the gene sequence is applied to the sheet. If the gene is expressed, the probe will hybridize with a specific mRNA molecule and a black band will appear on an Xray film. Other blotting techniques for detecting gene expression include '''Southern Blot''', in which mRNAs in a cell are reverse transcribed to their complementary DNA (cDNA) before being hybridized with gene-specific oligo-nucleotide probes. In a Western Blot experiment, the protein product (instead of the mRNA intermediate) of a gene is probed using antibodies (instead of the oligonucleotide probes).		Traditionally, gene expressions are studied one gene at a time using blotting techniques. For example, in a '''Northern Blot''' experiment ('''''Figure 4a'''''), the whole messenger RNA (mRNA) content of a cell is extracted and loaded on a solid gel slab. Different mRNA molecules are then separated using electrophoresis and transferred to a nitrocellulose sheet. To identify if a gene is expressed, a radioactively (or fluorescently) labeled oligonucleotide probe that is specific to the gene sequence is applied to the sheet. If the gene is expressed, the probe will hybridize with a specific mRNA molecule and a black band will appear on an Xray film. Other blotting techniques for detecting gene expression include '''Southern Blot''', in which mRNAs in a cell are reverse transcribed to their complementary DNA (cDNA) before being hybridized with gene-specific oligo-nucleotide probes. In a Western Blot experiment, the protein product (instead of the mRNA intermediate) of a gene is probed using antibodies (instead of the oligonucleotide probes).

	[[File:Bio_202_fig_4.jpg\|thumb\|frameless\|right\|'''Figure 4.''' <sub>'''a)''' Results from four individual Northern blots examining four different genes and measuring mRNA production over time, as indicated. b) Results from a series of microarrays for the same four genes of interest. Note the color scale on the bottom of '''b)''', where bright green indicates a 20fold repression and bright red indicates a 20fold		[[File:Bio_202_fig_4.jpg\|thumb\|frameless\|right\|'''Figure 4''' <sub>'''a)''' Results from four individual Northern blots examining four different genes and measuring mRNA production over time, as indicated. b) Results from a series of microarrays for the same four genes of interest. Note the color scale on the bottom of '''b)''', where bright green indicates a 20fold repression and bright red indicates a 20fold
	induction. Black indicates no change in transcription.</sub>]]		induction. Black indicates no change in transcription.</sub>]]
	After the genomic revolution since 1990s, it became possible to study the expression of all genes		After the genomic revolution since 1990s, it became possible to study the expression of all genes

imported>Dphilli at 02:39, 7 August 2013

2013-08-07T02:39:12Z

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	'''Question B:''' Now consider a culture where one yeast population (the experimental group) is grown in a solution where the amount of glucose decreases over time to zero, while a second population (the control group) is grown in flask where the Glucose concentration remains nearly constant. The researcher then extracts mRNA from the yeast cells to creates red-dyed-DNA for the experimental strain and green dye for cDNA made from the control strain. The dye-containing cDNA is then allowed to combine with a microarray containing spots with complementary DNA for a cytochrome C gene and a tRNA synthesase Gene. The expression levels in the control group are assumed to remain constant for both genes but the expression levels for the experimental group change according to the graph in '''Figure 5'''. In each situation, predict what color the two spots will show.		'''Question B:''' Now consider a culture where one yeast population (the experimental group) is grown in a solution where the amount of glucose decreases over time to zero, while a second population (the control group) is grown in flask where the Glucose concentration remains nearly constant. The researcher then extracts mRNA from the yeast cells to creates red-dyed-DNA for the experimental strain and green dye for cDNA made from the control strain. The dye-containing cDNA is then allowed to combine with a microarray containing spots with complementary DNA for a cytochrome C gene and a tRNA synthesase Gene. The expression levels in the control group are assumed to remain constant for both genes but the expression levels for the experimental group change according to the graph in '''Figure 5'''. In each situation, predict what color the two spots will show.


	[[File:Bio_202_4.jpg\|thumb\|frameless\|90px\|'''Table 1 (Question C)''']]		[[File:Bio_202_4.jpg\|thumb\|frameless\|90px\|'''Table 1 (Question C)''']]
			[[File:Bio_202_fig_2.jpg\|thumb\|frameless\|'''Figure 2''' <sub>The red and green cDNAs are mixed, placed on the same chip, covered by a glass coverslip, and incubated overnight with the DNA microarray. During this time, fluorescent cDNA will adhere to the appropriate spots on the glass slide. Later, under the bright light of a scanner, the spots will glow red or green, depending on which cDNA is bound to them and will glow yellow if similar amounts of both cDNA are bound to them. This glow can be quantified.</sub>.]]
			[[File:Bio_202_5.jpg\|thumb\|frameless\|60px\|'''Table 2 (Question D)''']]
	<big>'''2. Examining an Array Image'''</big>		<big>'''2. Examining an Array Image'''</big>
	[[File:Bio_202_fig_2.jpg\|thumb\|frameless\|'''Figure 2''' <sub>The red and green cDNAs are mixed, placed on the same chip, covered by a glass coverslip, and incubated overnight with the DNA microarray. During this time, fluorescent cDNA will adhere to the appropriate spots on the glass slide. Later, under the bright light of a scanner, the spots will glow red or green, depending on which cDNA is bound to them and will glow yellow if similar amounts of both cDNA are bound to them. This glow can be quantified.</sub>.]]
	[[File:Bio_202_fig_3.jpg\|thumb\|frameless\|'''Figure 3''' <sub>The first ten rows, 800 genes altogether, from one microarray used in the yeast starvation experiment we will discuss today. The spots appear either red for cDNA that was over-expressed in the glucose deprived experimental cells, green if that gene was under-expressed, yellow if the gene was equally expressed in both cells, or colorless if the gene was not expressed in either</sub>.]]		[[File:Bio_202_fig_3.jpg\|thumb\|frameless\|'''Figure 3''' <sub>The first ten rows, 800 genes altogether, from one microarray used in the yeast starvation experiment we will discuss today. The spots appear either red for cDNA that was over-expressed in the glucose deprived experimental cells, green if that gene was under-expressed, yellow if the gene was equally expressed in both cells, or colorless if the gene was not expressed in either</sub>.]]

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	The first line of the chart below is not from the microarray, it is simply a measurement of colony growth taken by shining light through the yeast colony as the cells multiplied. The second line is a measure of the ever-dropping nutrient concentration in the medium.		The first line of the chart below is not from the microarray, it is simply a measurement of colony growth taken by shining light through the yeast colony as the cells multiplied. The second line is a measure of the ever-dropping nutrient concentration in the medium.

	~~[[File:Bio_202_5.jpg\|thumb\|frameless\|60px\|'''Table 2 (Question D)''']]~~
	The third and fourth lines quantify the color of a single microarray spot (in this case, the gene NUP120) over time. For each time point, calculate the ratio of color intensity (red / green) and the base-two-logarithm of that ratio [ log<sub>2</sub>(red/green) ] . The first has been done for you as an example.		The third and fourth lines quantify the color of a single microarray spot (in this case, the gene NUP120) over time. For each time point, calculate the ratio of color intensity (red / green) and the base-two-logarithm of that ratio [ log<sub>2</sub>(red/green) ] . The first has been done for you as an example.

imported>Dphilli at 02:28, 7 August 2013

2013-08-07T02:28:59Z

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	'''Question L:''' Much of the previous questions in this lab were just preliminary work leading up to one		'''Question L:''' Much of the previous questions in this lab were just preliminary work leading up to one
	big question: ''Which genes form interactive networks?'' Let us now attempt to answer that! '''Table 5'''		big question: ''Which genes form interactive networks?'' Let us now attempt to answer that! '''Table 5'''
	showed the behavior of yeast genes under glucose deprivation. The correlation data (Pearson's correlation, '''''r''''') can are interpreted in '''Table 7''' as distances. The Table 7 data shows large values for disparate genes and approaches 0 for the ideal case where two genes are expressed in exactly the same levels in varying conditions. This then can be graphically shown as a dendrogram (tree) where similar expression gives close tree-distance. Three genes: NUP 145, COX4, and MLP1 still need to be placed on the dendrogram. Using the pairwise distances from Table 7, place these three genes in the proper location within the tree.		showed the behavior of yeast genes under glucose deprivation. The correlation data (Pearson's correlation, '''''r''''') can are interpreted in '''Table 7''' as distances. The Table 7 data shows large values for disparate genes and approaches 0 for the ideal case where two genes are expressed in exactly the same levels in varying conditions. This then can be graphically shown as a dendrogram (tree) where similar expression gives close tree-distance. Three genes: NUP 145, COX4, and MLP1 still need to be placed on the dendrogram. Using the pairwise distances from '''Table 7''', place these three genes in the proper location within the tree.

imported>Dphilli at 17:22, 6 August 2013

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	[[File:Bio_202_11.jpg\|thumb\|frameless\|'''Table 6 (Question K)''']]		[[File:Bio_202_11.jpg\|thumb\|frameless\|90px\|'''Table 6 (Question K)''']]
	<big>'''5. Database Search'''</big>		<big>'''5. Database Search'''</big>

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	: ''*For example, the DeRisi experiment found that the fold increase of TPS2 was 1.11, 1.15, 1.19, 2.04, 1.96, 4, 2.27. From this one can calculate the log<sub>2</sub> values filled into the first row of '''Table 6'''.''		: ''*For example, the DeRisi experiment found that the fold increase of TPS2 was 1.11, 1.15, 1.19, 2.04, 1.96, 4, 2.27. From this one can calculate the log<sub>2</sub> values filled into the first row of '''Table 6'''.''

	[[File:Bio_202_13.jpg\|right\|thumb\|frameless\|'''Figure 7''' <sub>This dendrogram takes the numerical distance values from Table 7 and displays them as visual distances.</sub>]]		[[File:Bio_202_13.jpg\|right\|thumb\|frameless\|90px\|'''Figure 7''' <sub>This dendrogram takes the numerical distance values from Table 7 and displays them as visual distances.</sub>]]


	<big>'''6. Distance Matrix and Clustering Dendrogram'''</big>		<big>'''6. Distance Matrix and Clustering Dendrogram'''</big>
	[[File:Bio_202_12.jpg\|thumb\|frameless\|'''Table 7''' <sub>This distance matrix was generated from the correlation data in '''''Table 5'''''.</sub>]]		[[File:Bio_202_12.jpg\|thumb\|frameless\|90px\|'''Table 7''' <sub>This distance matrix was generated from the correlation data in '''''Table 5'''''.</sub>]]

	'''Question L:''' Much of the previous questions in this lab were just preliminary work leading up to one		'''Question L:''' Much of the previous questions in this lab were just preliminary work leading up to one

imported>Dphilli at 17:18, 6 August 2013

2013-08-06T17:18:26Z

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	*Excel easily measures correlation and Pearson's r. Like most calculations in Excel, you simply click on an empty cell, type "=", write a formula, and indicate what range of cells you wish to perform the calculation on.		*Excel easily measures correlation and Pearson's r. Like most calculations in Excel, you simply click on an empty cell, type "=", write a formula, and indicate what range of cells you wish to perform the calculation on.

	[[File:Bio_202_8.jpg\|thumb\|framed\|center]]		[[File:Bio_202_8.jpg\|thumb\|framed\|100px\|center]]
	The above example was created by clicking on Cell J3 and entering the formula:		The above example was created by clicking on Cell J3 and entering the formula:
	'''=CORREL(B1:H1,B2:H2 )'''		'''=CORREL(B1:H1,B2:H2 )'''

imported>Dphilli at 17:15, 6 August 2013

2013-08-06T17:15:09Z

← Older revision		Revision as of 17:15, 6 August 2013
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	The first line of the chart below is not from the microarray, it is simply a measurement of colony growth taken by shining light through the yeast colony as the cells multiplied. The second line is a measure of the ever-dropping nutrient concentration in the medium.		The first line of the chart below is not from the microarray, it is simply a measurement of colony growth taken by shining light through the yeast colony as the cells multiplied. The second line is a measure of the ever-dropping nutrient concentration in the medium.

	[[File:Bio_202_5.jpg\|thumb\|frameless\|'''Table 2 (Question D)''']]		[[File:Bio_202_5.jpg\|thumb\|frameless\|60px\|'''Table 2 (Question D)''']]
	The third and fourth lines quantify the color of a single microarray spot (in this case, the gene NUP120) over time. For each time point, calculate the ratio of color intensity (red / green) and the base-two-logarithm of that ratio [ log<sub>2</sub>(red/green) ] . The first has been done for you as an example.		The third and fourth lines quantify the color of a single microarray spot (in this case, the gene NUP120) over time. For each time point, calculate the ratio of color intensity (red / green) and the base-two-logarithm of that ratio [ log<sub>2</sub>(red/green) ] . The first has been done for you as an example.

imported>Dphilli at 17:13, 6 August 2013

2013-08-06T17:13:14Z

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	==Procedures==		==Procedures==

	[[File:Bio_202_1.jpg\|thumb\|frameless\|'''Question A''']]		[[File:Bio_202_1.jpg\|thumb\|frameless\|85px\|'''Question A''']]
	<big>'''1. Microarray Color Prediction'''</big>		<big>'''1. Microarray Color Prediction'''</big>

	'''Question A:''' Consider a microarray with only two spots, one for Gene A and one for Gene B. A researcher cultures two yeast strains in Glucose solution, extracts mRNA from the cells, creates cDNA with either red dye (for the experimental strain) or green dye (for the control strain). The dye containing cDNA is then allowed to combine with a microarray containing spots with complementary DNA for Gene A and Gene B. In each situation below, predict what color the two spots will show. The first answer is filled in for you as an example.		'''Question A:''' Consider a microarray with only two spots, one for Gene A and one for Gene B. A researcher cultures two yeast strains in Glucose solution, extracts mRNA from the cells, creates cDNA with either red dye (for the experimental strain) or green dye (for the control strain). The dye containing cDNA is then allowed to combine with a microarray containing spots with complementary DNA for Gene A and Gene B. In each situation below, predict what color the two spots will show. The first answer is filled in for you as an example.

	[[File:Bio_202_3.jpg\|thumb\|frameless\|~~left~~\|'''Question B''']]		[[File:Bio_202_3.jpg\|thumb\|frameless\|right\|90px\|'''Question B''']]
	[[File:Bio_202_2.jpg\|thumb\|frameless\|left\|'''Figure 5'''. (from Cambell, p. 46)]]		[[File:Bio_202_2.jpg\|thumb\|frameless\|left\|100px\|'''Figure 5'''. (from Cambell, p. 46)]]

	'''Question B:''' Now consider a culture where one yeast population (the experimental group) is grown in a solution where the amount of glucose decreases over time to zero, while a second population (the control group) is grown in flask where the Glucose concentration remains nearly constant. The researcher then extracts mRNA from the yeast cells to creates red-dyed-DNA for the experimental strain and green dye for cDNA made from the control strain. The dye-containing cDNA is then allowed to combine with a microarray containing spots with complementary DNA for a cytochrome C gene and a tRNA synthesase Gene. The expression levels in the control group are assumed to remain constant for both genes but the expression levels for the experimental group change according to the graph in '''Figure 5'''. In each situation, predict what color the two spots will show.		'''Question B:''' Now consider a culture where one yeast population (the experimental group) is grown in a solution where the amount of glucose decreases over time to zero, while a second population (the control group) is grown in flask where the Glucose concentration remains nearly constant. The researcher then extracts mRNA from the yeast cells to creates red-dyed-DNA for the experimental strain and green dye for cDNA made from the control strain. The dye-containing cDNA is then allowed to combine with a microarray containing spots with complementary DNA for a cytochrome C gene and a tRNA synthesase Gene. The expression levels in the control group are assumed to remain constant for both genes but the expression levels for the experimental group change according to the graph in '''Figure 5'''. In each situation, predict what color the two spots will show.


			[[File:Bio_202_4.jpg\|thumb\|frameless\|90px\|'''Table 1 (Question C)''']]
	<big>'''2. Examining an Array Image'''</big>		<big>'''2. Examining an Array Image'''</big>
	~~[[File:Bio_202_4.jpg\|thumb\|frameless\|'''Table 1 (Question C)''']]~~
	[[File:Bio_202_fig_2.jpg\|thumb\|frameless\|'''Figure 2''' <sub>The red and green cDNAs are mixed, placed on the same chip, covered by a glass coverslip, and incubated overnight with the DNA microarray. During this time, fluorescent cDNA will adhere to the appropriate spots on the glass slide. Later, under the bright light of a scanner, the spots will glow red or green, depending on which cDNA is bound to them and will glow yellow if similar amounts of both cDNA are bound to them. This glow can be quantified.</sub>.]]		[[File:Bio_202_fig_2.jpg\|thumb\|frameless\|'''Figure 2''' <sub>The red and green cDNAs are mixed, placed on the same chip, covered by a glass coverslip, and incubated overnight with the DNA microarray. During this time, fluorescent cDNA will adhere to the appropriate spots on the glass slide. Later, under the bright light of a scanner, the spots will glow red or green, depending on which cDNA is bound to them and will glow yellow if similar amounts of both cDNA are bound to them. This glow can be quantified.</sub>.]]
	[[File:Bio_202_fig_3.jpg\|thumb\|frameless\|'''Figure 3''' <sub>The first ten rows, 800 genes altogether, from one microarray used in the yeast starvation experiment we will discuss today. The spots appear either red for cDNA that was over-expressed in the glucose deprived experimental cells, green if that gene was under-expressed, yellow if the gene was equally expressed in both cells, or colorless if the gene was not expressed in either</sub>.]]		[[File:Bio_202_fig_3.jpg\|thumb\|frameless\|'''Figure 3''' <sub>The first ten rows, 800 genes altogether, from one microarray used in the yeast starvation experiment we will discuss today. The spots appear either red for cDNA that was over-expressed in the glucose deprived experimental cells, green if that gene was under-expressed, yellow if the gene was equally expressed in both cells, or colorless if the gene was not expressed in either</sub>.]]

imported>Dphilli at 17:03, 6 August 2013

2013-08-06T17:03:26Z

← Older revision		Revision as of 17:03, 6 August 2013
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	<span style="color: Seagreen;font-weight:bold;font-size:large;">Exploration of Yeast Gene Expression Using Bioinformatics<span>		<span style="color: Seagreen;font-weight:bold;font-size:large;">Exploration of Yeast Gene Expression Using Bioinformatics<span>
	[[File:Bio_202_front.jpg\|~~right~~\|~~frameless~~]]		[[File:Bio_202_front.jpg\|frame\|none\|alt=Alt text]]

	==Objectives==		==Objectives==