20.109(S07): cDNA synthesis and microarray - Revision history

Nkuldell at 19:19, 20 April 2007

Nkuldell — Fri, 20 Apr 2007 19:19:08 GMT

Nzimm: /* Normalization */

Nzimm — Thu, 19 Apr 2007 18:28:03 GMT

Normalization

←Older revision		Revision as of 18:28, 19 April 2007
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	#Use the mean signal intensities (data in Columns B and C) from the Test Array to calculate the average intensity for the green and red signals. What is k?		#Use the mean signal intensities (data in Columns B and C) from the Test Array to calculate the average intensity for the green and red signals. What is k?
-	#Now use the median signal intensity (data in Columns G and H) to calculate k. Is there a difference when you calculate k using the mean and the median signal intensities?	+	#Now use the median signal intensity (data in Columns D and E) to calculate k. Is there a difference when you calculate k using the mean and the median signal intensities?
		+
	====Background correction====		====Background correction====

Nkuldell: /* Part 1: cDNA synthesis */

Nkuldell — Thu, 19 Apr 2007 17:59:21 GMT

Part 1: cDNA synthesis

←Older revision		Revision as of 17:59, 19 April 2007
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	\|}		\|}
	#Heat the annealing reactions to 80°C for 10 minutes then place the tubes on ice for 2 minutes.		#Heat the annealing reactions to 80°C for 10 minutes then place the tubes on ice for 2 minutes.
-	#Microfuge the tubes briefly to spin any condensation or droplets down to the bottom of the tube then add 1 ul of "Superase," ~~and~~ RNase inhibitor (Vial 4) and 8 ul of cDNA synthesis cocktail. Because reverse transcriptase is an unstable enzyme, this cocktail must be prepared just before use. The teaching faculty will prepare some for you when you are ready for it.	+	#Microfuge the tubes briefly to spin any condensation or droplets down to the bottom of the tube then add 1 ul of "Superase," an RNase inhibitor (Vial 4), and 8 ul of cDNA synthesis cocktail. Because reverse transcriptase is an unstable enzyme, this cocktail must be prepared just before use. The teaching faculty will prepare some for you when you are ready for it.
	#Pipet the contents of your tubes up and down to gently mix, then incubate the cDNA synthesis reactions at 42° for 1.5 hours. During this time, work with the sample array data file that is available (see Part 2 of today’s protocol).		#Pipet the contents of your tubes up and down to gently mix, then incubate the cDNA synthesis reactions at 42° for 1.5 hours. During this time, work with the sample array data file that is available (see Part 2 of today’s protocol).
	#Microfuge the tubes briefly then add 3.5 ul of 0.5M NaOH/0.5M EDTA to each tube and pipet up and down to mix. Heat to 65°C for 10 minutes. This step will denature your RNA/cDNA hybrids and degrade the RNA.		#Microfuge the tubes briefly then add 3.5 ul of 0.5M NaOH/0.5M EDTA to each tube and pipet up and down to mix. Heat to 65°C for 10 minutes. This step will denature your RNA/cDNA hybrids and degrade the RNA.

Nkuldell at 17:58, 19 April 2007

Nkuldell — Thu, 19 Apr 2007 17:58:48 GMT

←Older revision		Revision as of 17:58, 19 April 2007
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	\|}		\|}
	#Heat the annealing reactions to 80°C for 10 minutes then place the tubes on ice for 2 minutes.		#Heat the annealing reactions to 80°C for 10 minutes then place the tubes on ice for 2 minutes.
-	#Microfuge the tubes briefly to spin any condensation or droplets down to the bottom of the tube then add 9 ul of cDNA synthesis cocktail. Because reverse transcriptase is an unstable enzyme, this cocktail must be prepared just before use. The teaching faculty will prepare some for you when you are ready for it.	+	#Microfuge the tubes briefly to spin any condensation or droplets down to the bottom of the tube then add 1 ul of "Superase," and RNase inhibitor (Vial 4) and 8 ul of cDNA synthesis cocktail. Because reverse transcriptase is an unstable enzyme, this cocktail must be prepared just before use. The teaching faculty will prepare some for you when you are ready for it.
	#Pipet the contents of your tubes up and down to gently mix, then incubate the cDNA synthesis reactions at 42° for 1.5 hours. During this time, work with the sample array data file that is available (see Part 2 of today’s protocol).		#Pipet the contents of your tubes up and down to gently mix, then incubate the cDNA synthesis reactions at 42° for 1.5 hours. During this time, work with the sample array data file that is available (see Part 2 of today’s protocol).
	#Microfuge the tubes briefly then add 3.5 ul of 0.5M NaOH/0.5M EDTA to each tube and pipet up and down to mix. Heat to 65°C for 10 minutes. This step will denature your RNA/cDNA hybrids and degrade the RNA.		#Microfuge the tubes briefly then add 3.5 ul of 0.5M NaOH/0.5M EDTA to each tube and pipet up and down to mix. Heat to 65°C for 10 minutes. This step will denature your RNA/cDNA hybrids and degrade the RNA.

Jszab: /* To hybridize the arrays */ change hte for the.

Jszab — Tue, 17 Apr 2007 20:52:02 GMT

To hybridize the arrays: change hte for the.

←Older revision		Revision as of 20:52, 17 April 2007
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	#Heat your hybridization solutions to 80° for 10 minutes then cool them to room temperature. During this time you will be shown how to assemble the hybridization chambers.		#Heat your hybridization solutions to 80° for 10 minutes then cool them to room temperature. During this time you will be shown how to assemble the hybridization chambers.
	#Load your samples into the hybridization chambers as follows:		#Load your samples into the hybridization chambers as follows:
-	#*open the gasket and place the "Agilent" sticker facing up in the rectangular side of ~~hte~~ holder.	+	#*open the gasket and place the "Agilent" sticker facing up in the rectangular side of the holder.
	#*Pipet 200 ul of the hyb solution into place 1 or place 2 (see figure)[[Image:Macintosh HD-Users-nkuldell-Desktop-agilentarray.png]]		#*Pipet 200 ul of the hyb solution into place 1 or place 2 (see figure)[[Image:Macintosh HD-Users-nkuldell-Desktop-agilentarray.png]]
	#*Place the microarray <b><font color = red> "agilent" sticker down</font color> </b> onto the hybridization solution that's in the gasket.		#*Place the microarray <b><font color = red> "agilent" sticker down</font color> </b> onto the hybridization solution that's in the gasket.

Nkuldell: /* Part 2: practice array data analysis */

Nkuldell — Sat, 14 Apr 2007 11:12:52 GMT

Part 2: practice array data analysis

←Older revision		Revision as of 11:12, 14 April 2007
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	===Part 2: practice array data analysis===		===Part 2: practice array data analysis===
-	Data for these exercises should be downloaded from [[Media:Macintosh HD-Users-nkuldell-Desktop-PracticeData.xls\| this link]]	+	Data for these exercises should be downloaded from [[Media:Macintosh HD-Users-nkuldell-Desktop-PracticeData.xls\| this link]]. If you finish this exercise or if there is a break in your work you should also examine and/or scan and/or photograph the results of your spot tests from last time, when you were looking for phenotypes associated with the SAGA-deletion strains you have constructed.

	===Small data set===		===Small data set===

Nkuldell: /* Here’s what will happen tomorrow: */

Nkuldell — Sat, 14 Apr 2007 11:11:26 GMT

Here’s what will happen tomorrow:

←Older revision		Revision as of 11:11, 14 April 2007
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	*After 4 hours, the unbound Cy3 and Cy5 agents will be washed off your arrays. The wash steps will be 2X SSC/0.0016% Triton X-100 at 65°C for 10 minutes, 2X SSC/0.0016% Triton X-100 at room temperature for 5 minutes and 0.2X SSC/0.00016% Triton X-100 at room temperature for 10 minutes. The slides will be dried very quickly using Nitrogen gas to blow off any water droplets, then scanned in the Agilent scanner that is available in the BioMicroCenter. The data from your array will be available for you to analyze next time.		*After 4 hours, the unbound Cy3 and Cy5 agents will be washed off your arrays. The wash steps will be 2X SSC/0.0016% Triton X-100 at 65°C for 10 minutes, 2X SSC/0.0016% Triton X-100 at room temperature for 5 minutes and 0.2X SSC/0.00016% Triton X-100 at room temperature for 10 minutes. The slides will be dried very quickly using Nitrogen gas to blow off any water droplets, then scanned in the Agilent scanner that is available in the BioMicroCenter. The data from your array will be available for you to analyze next time.

-	~~Before you leave today you should also be sure to examine and/or scan the results of your spot tests for phenotypes associated with the SAGA-deletion strains you have constructed.~~

	DONE!		DONE!
		+
	==For next time==		==For next time==
	#Prepare a figure for phenotype plates. This figure should include a title and legend as before and should contain sufficient information that a reader can get the idea without reading the article that will eventually accompany this figure.		#Prepare a figure for phenotype plates. This figure should include a title and legend as before and should contain sufficient information that a reader can get the idea without reading the article that will eventually accompany this figure.

Nkuldell: /* to hybridize the arrays */

Nkuldell — Sat, 14 Apr 2007 11:10:37 GMT

to hybridize the arrays

←Older revision		Revision as of 11:10, 14 April 2007
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	*Each array has a barcode printed on some stickers on one end of each slide. <b>The array is printed on the side of the slide that says “Agilent.” </b> If you try to hybridize your cDNA to the numbered side of the slide, there will be no array there to bind. <br>		*Each array has a barcode printed on some stickers on one end of each slide. <b>The array is printed on the side of the slide that says “Agilent.” </b> If you try to hybridize your cDNA to the numbered side of the slide, there will be no array there to bind. <br>

-	====to hybridize the arrays====	+	====To hybridize the arrays====
	#Begin by mixing the cDNA pools you have synthesized into one eppendorf tube. The total volume should be 57 ul. Add 43 ul of H2O and 100 ul of 2X Hybridization Buffer. Pipet up and down several times to mix the contents.		#Begin by mixing the cDNA pools you have synthesized into one eppendorf tube. The total volume should be 57 ul. Add 43 ul of H2O and 100 ul of 2X Hybridization Buffer. Pipet up and down several times to mix the contents.
	#Heat your hybridization solutions to 80° for 10 minutes then cool them to room temperature. During this time you will be shown how to assemble the hybridization chambers.		#Heat your hybridization solutions to 80° for 10 minutes then cool them to room temperature. During this time you will be shown how to assemble the hybridization chambers.
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	#*Tighten and tape them with your colored tape.		#*Tighten and tape them with your colored tape.

-	When every group is ready, we will walk the arrays over to the BioMicroCenter in Building 68 to put them in the 60°C hybridization oven.	+	When every group is ready, we will walk the arrays over to the BioMicroCenter in Building 68 to put them in the 60°C hybridization oven.

	====Here’s what will happen tomorrow:====		====Here’s what will happen tomorrow:====

Nkuldell: /* intensity ratios */

Nkuldell — Sat, 14 Apr 2007 11:09:56 GMT

intensity ratios

←Older revision		Revision as of 11:09, 14 April 2007
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	#Find one green background measurement that is considerably different from the average. Is the red background measurement also different? How could you explain this?		#Find one green background measurement that is considerably different from the average. Is the red background measurement also different? How could you explain this?
	#Insert two new columns after the background signal columns and calculate the “background corrected” values for the green and red signals. These corrected values are determined by subtracting the background measurement for each spot from the signal measurement.		#Insert two new columns after the background signal columns and calculate the “background corrected” values for the green and red signals. These corrected values are determined by subtracting the background measurement for each spot from the signal measurement.
-	====~~intensity~~ ratios====	+	====Intensity ratios====
	So far you’ve seen that microarray data must be normalized to correct for Cy3 and Cy5 differences as well as “background subtracted” to correct for artifacts on the slide. Recall that microarray experiments are designed to simultaneously compare the expression of many genes in two samples. The corrected intensities can be expressed as a ratio between the corrected signals for the two samples (Green/Red). A ratio of 4 means 4-fold gene induction and a ratio of 0.25 means four-fold repression of that gene.		So far you’ve seen that microarray data must be normalized to correct for Cy3 and Cy5 differences as well as “background subtracted” to correct for artifacts on the slide. Recall that microarray experiments are designed to simultaneously compare the expression of many genes in two samples. The corrected intensities can be expressed as a ratio between the corrected signals for the two samples (Green/Red). A ratio of 4 means 4-fold gene induction and a ratio of 0.25 means four-fold repression of that gene.

Nkuldell: /* Part 2: practice array data analysis */

Nkuldell — Sat, 14 Apr 2007 11:09:07 GMT

Part 2: practice array data analysis

←Older revision		Revision as of 11:09, 14 April 2007
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	===Part 2: practice array data analysis===		===Part 2: practice array data analysis===
-	Data for these exercises should be downloaded from ~~this link:~~ [[~~Image~~:Macintosh HD-Users-nkuldell-Desktop-PracticeData.xls]]	+	Data for these exercises should be downloaded from [[Media:Macintosh HD-Users-nkuldell-Desktop-PracticeData.xls\| this link]]

	===Small data set===		===Small data set===

←Older revision		Revision as of 19:19, 20 April 2007
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	===Small data set===		===Small data set===

-	Begin with the sheet called “Sample Data” which contains all the data for 16 of the 22,000 spots on the tested array. This sheet will familiarize you with the headings and entries you can expect to see when you examine your own data. Some important landmarks are	+	Begin with the sheet called “Sample Data” which contains all the data for 21 of the 22,000 spots on the tested array. This sheet will familiarize you with the headings and entries you can expect to see when you examine your own data. Some important landmarks are
	*Columns C and D give the address for each gene on the array		*Columns C and D give the address for each gene on the array
	*Columns J and K list the gene name associated with each spot		*Columns J and K list the gene name associated with each spot
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	#What is the systematic name for SLG1?		#What is the systematic name for SLG1?
	#What is the cellular function for SNF2?		#What is the cellular function for SNF2?
-	#The array annotates FUN11 as “function unknown.” Does this agree with the SGD annotation?	+	#The array annotates FUN11 as “function unknown.” Does this agree with the [http://www.yeastgenome.org/\| SGD] annotation?
-	#Where is PSD1 localized? does this agree with the SGD annotation?	+	#Where is the PSD1 gene product localized in the cell? does this agree with the SGD annotation for PSD1?

-	To look at the signal intensities for these 16 spots, you should scroll right. Column AF reports the signal from the green fluorescent molecule Cy3 and Column AG reports the signal from the red fluorescent molecule Cy5. Use the “Format” menu to convert the values in these columns from scientific notation to numbers with no decimal places, and then answer the following questions.	+	To look at the signal intensities for these 21 spots, you should scroll right. Column AF reports the signal from the green fluorescent molecule Cy3 and Column AG reports the signal from the red fluorescent molecule Cy5. Use the “Format” menu to convert the values in these columns from scientific notation to numbers with no decimal places, and then answer the following questions.

	#What are the green and red signal intensities for MFA2?		#What are the green and red signal intensities for MFA2?
	#What genes give the highest and lowest values in Column AF?		#What genes give the highest and lowest values in Column AF?
	#Are these the same genes that give the highest and lowest values in Column AG?		#Are these the same genes that give the highest and lowest values in Column AG?
-	#What fraction of the ~~genes~~ listed have a larger value in Column AF than in Column AG?	+	#What fraction of the listed values have a larger value in Column AF than in Column AG?
	#Using the values in Column AF, find the mean and the median value for the three SDC25 signals. What does the agreement of the mean and median tell you about the three values?		#Using the values in Column AF, find the mean and the median value for the three SDC25 signals. What does the agreement of the mean and median tell you about the three values?
	#Find the mean and the median for all the values in Column AF and AG. What is the significance that the mean and the median values are not identical? What is the significance that the mean value for Column AF is not identical to the mean value for Column AG?		#Find the mean and the median for all the values in Column AF and AG. What is the significance that the mean and the median values are not identical? What is the significance that the mean value for Column AF is not identical to the mean value for Column AG?