20.109(S13):Preparing cells for analysis (Day4) - Revision history

AgiStachowiak at 14:46, 29 April 2013

AgiStachowiak — Mon, 29 Apr 2013 14:46:00 GMT

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-	<font color=red>~~currently in revision -- light~~ edits ~~coming~~ Monday morning</font color>	+	<font color=red>Done. Light edits completed Monday morning.</font color>

	==Introduction==		==Introduction==

AgiStachowiak: /* Cell Counting */

AgiStachowiak — Mon, 29 Apr 2013 14:43:31 GMT

Cell Counting

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	Your goal for this section will be to compare the effort required for, and the resulting accuracy of, manually counting live and dead cells vs. doing so by semi-automated image analysis. After you are done, you might consider under what conditions you might prefer one method or the other.		Your goal for this section will be to compare the effort required for, and the resulting accuracy of, manually counting live and dead cells vs. doing so by semi-automated image analysis. After you are done, you might consider under what conditions you might prefer one method or the other.

-	If you don't have many cells to look at, extra data is available on today's [[Talk:20.109%~~28S11~~%29:Preparing_cells_for_analysis_%28Day4%29 \| Talk]] page.	+	If you don't have many cells to look at, extra data is available on today's [[Talk:20.109%28S13%29:Preparing_cells_for_analysis_%28Day4%29 \| Talk]] page.

	#Open your live cell image (green filter) by selecting ''File'' → ''Open''		#Open your live cell image (green filter) by selecting ''File'' → ''Open''
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	#*One way to do this is by choosing ''Process'' → ''Binary'' → ''Make Binary''. However, you might find that clusters of cells are ‘read’ as a single cell.		#*One way to do this is by choosing ''Process'' → ''Binary'' → ''Make Binary''. However, you might find that clusters of cells are ‘read’ as a single cell.
	#*An improved method is to use'' Image'' → ''Adjust'' → ''Threshold''. You can set the upper- and lower-bound intensities that define objects in your image. Play with the intensity sliders until your cells are mostly filled in with a red colour, but not overlapping with other cells whenever possible.		#*An improved method is to use'' Image'' → ''Adjust'' → ''Threshold''. You can set the upper- and lower-bound intensities that define objects in your image. Play with the intensity sliders until your cells are mostly filled in with a red colour, but not overlapping with other cells whenever possible.
-	#Now you can count the objects. Choose ''Analyze'' → ''Analyze Particles'', and select ~~'''~~''Show~~'''~~'' →~~'''~~''Outlines~~'''~~'', '''Display Results''', '''Summarize''', and '''Record Stats'''. Also choose a reasonable area (not diameter!) range for objects that are cell-sized.	+	#Now you can count the objects. Choose ''Analyze'' → ''Analyze Particles'', and select ''Show'' →''Outlines'', ''Display Results'', ''Summarize'', and ''Record Stats''. Also choose a reasonable '''area''' (not diameter!) range for objects that are cell-sized.
	#Try to play around with this process for a bit – are there any further changes you can make so the automated algorithm is as good as your eye?		#Try to play around with this process for a bit – are there any further changes you can make so the automated algorithm is as good as your eye?
-	#Record your final results (manual and best automated) in your notebook, for each sample that you have data for. Don’t forget to also count the dead cells, and subtract this number from the live cells (since the filter we used shows both green and red cells at once).	+	#Record your final results (manual and best automated) in your notebook, for each sample that you have data for. Don’t forget to also manually count the dead cells, and subtract this number from the live cells (since the filter we used shows both green and red cells at once).

	=====Statistical Analysis=====		=====Statistical Analysis=====

AgiStachowiak: /* Part 5: Begin viability analysis (optional) */

AgiStachowiak — Mon, 29 Apr 2013 14:41:00 GMT

Part 5: Begin viability analysis (optional)

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	===Part 5: Begin viability analysis (optional)===		===Part 5: Begin viability analysis (optional)===

-	Starting today or as late as Day 6, you will use an image analysis program called ImageJ to quantify your live/dead data. This is offered free of charge by the NIH (National Institutes of Health). ~~To make the TA's job easier, please hand~~ in ~~your viability analysis and answers~~ to ~~the questions on Day 6, whether you do it earlier or not~~.	+	Starting today or as late as Day 6, you will use an image analysis program called ImageJ to quantify your live/dead data. This program is offered free of charge by the NIH (National Institutes of Health). Folks in W/F should feel free to practice with some T/R samples.

	=====Cell Counting=====		=====Cell Counting=====

AgiStachowiak: /* Part 4: RT reactions */

AgiStachowiak — Mon, 29 Apr 2013 14:39:30 GMT

Part 4: RT reactions

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	#Set up your reactions on a cold block as usual. You will prepare one reaction for each of your samples. Random hexamer primers will be used so that all (we hope) transcripts are amplified. This approach is more convenient than adding unique primers for each transcript of interest.		#Set up your reactions on a cold block as usual. You will prepare one reaction for each of your samples. Random hexamer primers will be used so that all (we hope) transcripts are amplified. This approach is more convenient than adding unique primers for each transcript of interest.
-	#From one of the shared stocks, pipet 22.5 μL of ~~Master Mix~~ into each of two well-labeled PCR tubes. The ~~Master~~ mix contains water, buffer, dNTPS, primers, and ~~and an enzyme mixture~~.	+	#From one of the shared stocks, pipet 22.5 μL of RT master mix into each of two well-labeled PCR tubes. The master mix contains water, buffer, dNTPS, primers, and reverse transcriptase.
	#Now you can add 7.5 μL of the appropriate RNA (or RNA and water as needed) to each tube.		#Now you can add 7.5 μL of the appropriate RNA (or RNA and water as needed) to each tube.
-	#The following thermal cycler program will be used: 20 min at 25 °C, 30 min at 42 °C (reverse transcription step), then cooling. After the RT is completed, the teaching faculty will store the samples in the freezer until next time.	+	#The following thermal cycler program will be used: 20 min at 25 °C, 30 min at 42 °C (reverse transcription step), and then cooling. After the RT is completed, the teaching faculty will store the samples in the freezer until next time.

-	<font color=purple>~~'''~~In whatever time remains today, you can continue discussion of your shared research idea with your partner, work on your Module 3 report~~, etc~~. The ~~easiest thing might be~~ to ~~get the analysis of~~ your ~~Day 3 data out of the way, as described~~ below.</font color>	+	<font color=purple>In whatever time remains today, you can continue discussion of your shared research idea with your partner or work on your Module 3 report. The steps to analyze your cell viability images are outlined below.</font color>

	===Part 5: Begin viability analysis (optional)===		===Part 5: Begin viability analysis (optional)===

AgiStachowiak: /* Part 4: RT reactions */

AgiStachowiak — Mon, 29 Apr 2013 14:36:56 GMT

Part 4: RT reactions

←Older revision		Revision as of 14:36, 29 April 2013
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	===Part 4: RT reactions===		===Part 4: RT reactions===

-	#Set up your reactions on a cold block as usual. You will prepare one reaction for each of your samples. Random hexamer primers will be used so that all (we hope) transcripts are amplified. This is more convenient than adding unique primers for each transcript of interest.	+	#Set up your reactions on a cold block as usual. You will prepare one reaction for each of your samples. Random hexamer primers will be used so that all (we hope) transcripts are amplified. This approach is more convenient than adding unique primers for each transcript of interest.
	#From one of the shared stocks, pipet 22.5 μL of Master Mix into each of two well-labeled PCR tubes. The Master mix contains water, buffer, dNTPS, primers, and and an enzyme mixture.		#From one of the shared stocks, pipet 22.5 μL of Master Mix into each of two well-labeled PCR tubes. The Master mix contains water, buffer, dNTPS, primers, and and an enzyme mixture.
	#Now you can add 7.5 μL of the appropriate RNA (or RNA and water as needed) to each tube.		#Now you can add 7.5 μL of the appropriate RNA (or RNA and water as needed) to each tube.

AgiStachowiak: /* Part 3: RNA isolation and measurement */

AgiStachowiak — Mon, 29 Apr 2013 14:34:54 GMT