20.109(S13):Transcript-level analysis (Day6) - Revision history

AgiStachowiak: /* Part 3: qPCR analysis */

2013-05-08T15:59:25Z

Part 3: qPCR analysis

←Older revision		Revision as of 15:59, 8 May 2013
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	#Finally, look briefly at your melt curves. As we discussed in lecture, the samples should each exhibit a single peak that can be attributed to the melting of the desired product. There may also be a small primer-dimer peak, about 10% of the main peak intensity. Do any of your samples have a substantial amount of non-specific (most likely primer-dimer) product?		#Finally, look briefly at your melt curves. As we discussed in lecture, the samples should each exhibit a single peak that can be attributed to the melting of the desired product. There may also be a small primer-dimer peak, about 10% of the main peak intensity. Do any of your samples have a substantial amount of non-specific (most likely primer-dimer) product?
	#*T/R: CN I curves all great. CN II curves: C1+2, G1+2 appear multi-product; C3+4 shifted to higher Tm; G3+4 slight shoulder. 18S curves: Double-peaked for almost everyone, with some very low (rows E+F) and others relatively high (A9, C11, D12).		#*T/R: CN I curves all great. CN II curves: C1+2, G1+2 appear multi-product; C3+4 shifted to higher Tm; G3+4 slight shoulder. 18S curves: Double-peaked for almost everyone, with some very low (rows E+F) and others relatively high (A9, C11, D12).
-	#*W/F: All three curve sets showed a fair amount of scatter in Tm, usually about 1 degree. I won't list them in their entirety, but for example for CN II, rows A, E, and F were around 84.5 and B, C around 83.5.	+	#*W/F: All three curve sets showed a fair amount of scatter in Tm, usually about 1 degree. I won't list them in their entirety, but for example for CN II, rows A, E, and F were around 84.5 and B, C around 83.5. The following samples had rather low peaks: D3+4, F3+4, G and H; B9+10, E9+10, G and H. As in T/R section, the 18S melt curves showed a secondary peak in many cases, though it was often low. The highest secondary peaks were in C11, D9, D12, and F10.
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AgiStachowiak: /* Part 3: qPCR analysis */

2013-05-08T15:56:08Z

Part 3: qPCR analysis

←Older revision		Revision as of 15:56, 8 May 2013
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	#Finally, look briefly at your melt curves. As we discussed in lecture, the samples should each exhibit a single peak that can be attributed to the melting of the desired product. There may also be a small primer-dimer peak, about 10% of the main peak intensity. Do any of your samples have a substantial amount of non-specific (most likely primer-dimer) product?		#Finally, look briefly at your melt curves. As we discussed in lecture, the samples should each exhibit a single peak that can be attributed to the melting of the desired product. There may also be a small primer-dimer peak, about 10% of the main peak intensity. Do any of your samples have a substantial amount of non-specific (most likely primer-dimer) product?
	#*T/R: CN I curves all great. CN II curves: C1+2, G1+2 appear multi-product; C3+4 shifted to higher Tm; G3+4 slight shoulder. 18S curves: Double-peaked for almost everyone, with some very low (rows E+F) and others relatively high (A9, C11, D12).		#*T/R: CN I curves all great. CN II curves: C1+2, G1+2 appear multi-product; C3+4 shifted to higher Tm; G3+4 slight shoulder. 18S curves: Double-peaked for almost everyone, with some very low (rows E+F) and others relatively high (A9, C11, D12).
-	#*W/F: ~~Comments coming by first thing Wed AM!~~	+	#*W/F: All three curve sets showed a fair amount of scatter in Tm, usually about 1 degree. I won't list them in their entirety, but for example for CN II, rows A, E, and F were around 84.5 and B, C around 83.5.
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AgiStachowiak: /* Part 3: qPCR analysis */

2013-05-08T13:50:20Z

Part 3: qPCR analysis

←Older revision		Revision as of 13:50, 8 May 2013
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	#Begin by looking at the CP values (and raw or threshold-processed curves if available). Comment on replicate agreement.		#Begin by looking at the CP values (and raw or threshold-processed curves if available). Comment on replicate agreement.
	#*T/R S13 notes: Sample B2 has an early slow rise, never really exponential, suggesting some kind of error/artifact. (Might just be a really high starting [cDNA].) Sample C3 had an odd dip down before coming back up. Samples C1+2, D1+2, and G1+2 seem to still be in the first plateau rather than curving up with the gene-specific noiseband, meaning that the crossing points are underestimated by maybe 1-2 cycles (C1+2 closer to 21, D1+2 to 20, G1+2 to 19) -- take the data with a grain of salt. Note that samples H3+4 (supposedly no template!) gave a slow-rise signal.		#*T/R S13 notes: Sample B2 has an early slow rise, never really exponential, suggesting some kind of error/artifact. (Might just be a really high starting [cDNA].) Sample C3 had an odd dip down before coming back up. Samples C1+2, D1+2, and G1+2 seem to still be in the first plateau rather than curving up with the gene-specific noiseband, meaning that the crossing points are underestimated by maybe 1-2 cycles (C1+2 closer to 21, D1+2 to 20, G1+2 to 19) -- take the data with a grain of salt. Note that samples H3+4 (supposedly no template!) gave a slow-rise signal.
-	#*W/F S13 notes: ~~Comments coming by first thing Wed AM!~~	+	#*W/F S13 notes: The following samples registered as having no signal to the machine: D3+4; F3+4, 7+8, 11+12; Row G; H4, 6-12. CNII: Sample H4 had an odd rise -- likely negative just as H1-H3 are. CN I: Samples B 7+8 have a late slow rise, are essentially negative for that gene. (Okay to use the high Cp in your calculations, just be aware.) D7+8 have a somewhat odd shape -- take with a grain of salt. E8 has a higher noise level, and the Cp may be misleading compared to well E7. Sample H5 has an odd shape, just an artifact. 18S: C11 has a somewhat odd shape, is less trustworthy compared to C12. Samples D11+12 have a late slow rise, are negative for that gene. (Okay to use the high Cp in your calculations, just be aware.)
	#Calculate the average and standard deviation of each of your 6 pairs of CP values.		#Calculate the average and standard deviation of each of your 6 pairs of CP values.
	#Based on your average CP values, calculate the change in gene expression if you consider one of your samples the control and the other the sample. You might want to start with the practice calculation in step 6.		#Based on your average CP values, calculate the change in gene expression if you consider one of your samples the control and the other the sample. You might want to start with the practice calculation in step 6.

AgiStachowiak: /* Part 3: qPCR analysis */

2013-05-07T16:46:55Z

Part 3: qPCR analysis

←Older revision		Revision as of 16:46, 7 May 2013
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	#On today's Talk page you will download your CP (also called C<sub>T</sub>) values. You will also be able to view section-wide melt curves and amplification curves per each primer set.		#On today's Talk page you will download your CP (also called C<sub>T</sub>) values. You will also be able to view section-wide melt curves and amplification curves per each primer set.
	#Begin by looking at the CP values (and raw or threshold-processed curves if available). Comment on replicate agreement.		#Begin by looking at the CP values (and raw or threshold-processed curves if available). Comment on replicate agreement.
-	#*T/R S13 notes: Sample B2 has an early slow rise, never really exponential, suggesting some kind of error/artifact. Sample G3 had an odd dip down before coming back up. ~~<font color=red>On~~ closer ~~inspection~~, a ~~couple~~ of ~~other CN II~~ samples ~~are oddly shaped -~~- ~~will post these wells later~~.~~</font color>~~	+	#*T/R S13 notes: Sample B2 has an early slow rise, never really exponential, suggesting some kind of error/artifact. (Might just be a really high starting [cDNA].) Sample C3 had an odd dip down before coming back up. Samples C1+2, D1+2, and G1+2 seem to still be in the first plateau rather than curving up with the gene-specific noiseband, meaning that the crossing points are underestimated by maybe 1-2 cycles (C1+2 closer to 21, D1+2 to 20, G1+2 to 19) -- take the data with a grain of salt. Note that samples H3+4 (supposedly no template!) gave a slow-rise signal.
	#*W/F S13 notes: Comments coming by first thing Wed AM!		#*W/F S13 notes: Comments coming by first thing Wed AM!
	#Calculate the average and standard deviation of each of your 6 pairs of CP values.		#Calculate the average and standard deviation of each of your 6 pairs of CP values.

AgiStachowiak: /* Part 3: qPCR analysis */

2013-05-06T19:33:54Z

Part 3: qPCR analysis

←Older revision		Revision as of 19:33, 6 May 2013
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	#On today's Talk page you will download your CP (also called C<sub>T</sub>) values. You will also be able to view section-wide melt curves and amplification curves per each primer set.		#On today's Talk page you will download your CP (also called C<sub>T</sub>) values. You will also be able to view section-wide melt curves and amplification curves per each primer set.
	#Begin by looking at the CP values (and raw or threshold-processed curves if available). Comment on replicate agreement.		#Begin by looking at the CP values (and raw or threshold-processed curves if available). Comment on replicate agreement.
-	#*T/R S13 notes: ~~[samples that were below detection limit~~, ~~etc~~.]	+	#*T/R S13 notes: Sample B2 has an early slow rise, never really exponential, suggesting some kind of error/artifact. Sample G3 had an odd dip down before coming back up. <font color=red>On closer inspection, a couple of other CN II samples are oddly shaped -- will post these wells later.</font color>
-	#*W/F S13 notes:	+	#*W/F S13 notes: Comments coming by first thing Wed AM!
	#Calculate the average and standard deviation of each of your 6 pairs of CP values.		#Calculate the average and standard deviation of each of your 6 pairs of CP values.
	#Based on your average CP values, calculate the change in gene expression if you consider one of your samples the control and the other the sample. You might want to start with the practice calculation in step 6.		#Based on your average CP values, calculate the change in gene expression if you consider one of your samples the control and the other the sample. You might want to start with the practice calculation in step 6.
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	#*You can see that in 2D culture, the additions of proline and ascorbate make only a small impact, if any, on CN II and CN I expression.		#*You can see that in 2D culture, the additions of proline and ascorbate make only a small impact, if any, on CN II and CN I expression.
	#Finally, look briefly at your melt curves. As we discussed in lecture, the samples should each exhibit a single peak that can be attributed to the melting of the desired product. There may also be a small primer-dimer peak, about 10% of the main peak intensity. Do any of your samples have a substantial amount of non-specific (most likely primer-dimer) product?		#Finally, look briefly at your melt curves. As we discussed in lecture, the samples should each exhibit a single peak that can be attributed to the melting of the desired product. There may also be a small primer-dimer peak, about 10% of the main peak intensity. Do any of your samples have a substantial amount of non-specific (most likely primer-dimer) product?
-	#*T/R:	+	#*T/R: CN I curves all great. CN II curves: C1+2, G1+2 appear multi-product; C3+4 shifted to higher Tm; G3+4 slight shoulder. 18S curves: Double-peaked for almost everyone, with some very low (rows E+F) and others relatively high (A9, C11, D12).
-	#*W/F:	+	#*W/F: Comments coming by first thing Wed AM!
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AgiStachowiak: /* Part 4: Cross-group research idea discussion */

2013-05-03T18:07:43Z

Part 4: Cross-group research idea discussion

←Older revision		Revision as of 18:07, 3 May 2013
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	===Part 4: Cross-group research idea discussion===		===Part 4: Cross-group research idea discussion===

-	You can complete this discussion today or outside of class, according to the pairings on today's [[Talk:20.109%~~28S11~~%29:Transcript-level_analysis_%28Day6%29 \| Talk]] page.	+	You can complete this discussion today or outside of class, according to the pairings on today's [[Talk:20.109%28S13%29:Transcript-level_analysis_%28Day6%29 \| Talk]] page.

	You should be on your way to becoming an expert on your research topic. You should have been reading and thinking a lot about it and you may feel <br>		You should be on your way to becoming an expert on your research topic. You should have been reading and thinking a lot about it and you may feel <br>

AgiStachowiak: /* Part 5: Continue ImageJ analysis (optional) */

2013-05-03T15:59:03Z

Part 5: Continue ImageJ analysis (optional)

←Older revision		Revision as of 15:59, 3 May 2013
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	===Part 5: Continue ImageJ analysis (optional)===		===Part 5: Continue ImageJ analysis (optional)===

-	~~In your notebook, you should comment~~ on your live/dead assay imaging analysis if you did not do so last time.	+	Work on your live/dead assay imaging analysis if you did not do so last time.

	==For next time==		==For next time==

AgiStachowiak: /* Part 3: qPCR analysis */

2013-05-03T15:56:10Z

Part 3: qPCR analysis

←Older revision		Revision as of 15:56, 3 May 2013
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	#On today's Talk page you will download your CP (also called C<sub>T</sub>) values. You will also be able to view section-wide melt curves and amplification curves per each primer set.		#On today's Talk page you will download your CP (also called C<sub>T</sub>) values. You will also be able to view section-wide melt curves and amplification curves per each primer set.
	#Begin by looking at the CP values (and raw or threshold-processed curves if available). Comment on replicate agreement.		#Begin by looking at the CP values (and raw or threshold-processed curves if available). Comment on replicate agreement.
-	#*T/R: The CP for wells D1, D2, E3, and E4 looks to be an artifact; the curve slowly rises rather than showing exponential amplification at any time. Wells D5, D6, E7, E8, E11, and E12 registered as containing no sample to the machine.	+	#*T/R S13 notes: [samples that were below detection limit, etc.]
-	~~#*<font color=red>New T/R~~ notes~~</font color>~~: Wells A1-2 Cp is probably closer to 22; wells B1-4 Cp is probably closer to 20; wells D3-4 Cp is closer to 19; wells E1-2 is closer to 17; wells G1-2 may be slightly shifted to a higher Cp than wells G3-4. Here I'm referring to by-gene Cps rather than all together.	+	#*W/F S13 notes:
-	~~#*W/F: C5 sample rose early and is oddly shaped so can't be trusted. A1, A3, C1-2, G3-4 registered as containing no sample to the machine (along with some others~~ that ~~I didn't note~~, ~~based on the number of missing Cp values and flat curves - I will check today or tomorrow morning~~.)	+
-	#~~<font color=red>New~~ W/F notes~~</font color>~~: Analysis was re-done excluded sample C3 (not C5, that's an error above!) due to its effect on the noise threshold. By-gene Cp values for 18S and CN I are unaffected, but by-gene values for CN II along with Cp all values for all three primer sets are affected. Samples B1and B3 show an initially slow rise, so the new Cp by-gene values are probably closer to 25-26 than what is listed. Using the original* noiseband for this data is probably best. Exhaustive list of samples registering no detectable cDNA (whether overall or gene-specific): A1, C1-8, G3-4,7-8,11-2. Well A3 has a very late and slow rise, indicating non-specific product only.	+
	#Calculate the average and standard deviation of each of your 6 pairs of CP values.		#Calculate the average and standard deviation of each of your 6 pairs of CP values.
	#Based on your average CP values, calculate the change in gene expression if you consider one of your samples the control and the other the sample. You might want to start with the practice calculation in step 6.		#Based on your average CP values, calculate the change in gene expression if you consider one of your samples the control and the other the sample. You might want to start with the practice calculation in step 6.
	#*For multi-group collaborations, you can consistently use one of the 4<sup>+</sup> samples as the control if appropriate.		#*For multi-group collaborations, you can consistently use one of the 4<sup>+</sup> samples as the control if appropriate.
	#*Note that the sample is subtracted ''from'' the control (to ultimately get the expression ratio of the sample relative to the control), not the other way around.		#*Note that the sample is subtracted ''from'' the control (to ultimately get the expression ratio of the sample relative to the control), not the other way around.
-	#~~Now~~ calculate the gene expression change relative to pure chondrocytes or pure stem cells. ~~The CP values are listed in the table below.~~	+	#You can also try to calculate the gene expression change relative to pure chondrocytes or pure stem cells. However, to give reliable results these samples should have been run in the same plate as yours with the same day's master mix. The CP values are listed in the table below.
-	~~#*Note: ideally~~, these samples should have been run in the same plate as yours with the same day's master mix. ~~However, we have found very good day-today consistency for CN I, with somewhat worse repeatability for CN II and 18S~~.	+
	#*Note that the stem cell CN II value may be somewhat off, due to combining two imperfect experiments. It may be closer to 22, or even a bit higher. (Which makes a big difference to the ratio!)		#*Note that the stem cell CN II value may be somewhat off, due to combining two imperfect experiments. It may be closer to 22, or even a bit higher. (Which makes a big difference to the ratio!)
	#CP values for extended (2D) chondrocyte culture in medium with or without both proline and ascorbate are also shown in the table below. As practice and a check on your ratio calculations above, you should try calculating the expression ratios for with vs. without chondrocytic media factors, with vs. chondrocytes, without vs. chondrocytes, and chondrocytes vs. stem cells. You can compare your answers to those in the table, and fix your analysis above if needed.		#CP values for extended (2D) chondrocyte culture in medium with or without both proline and ascorbate are also shown in the table below. As practice and a check on your ratio calculations above, you should try calculating the expression ratios for with vs. without chondrocytic media factors, with vs. chondrocytes, without vs. chondrocytes, and chondrocytes vs. stem cells. You can compare your answers to those in the table, and fix your analysis above if needed.
	#*You can see that in 2D culture, the additions of proline and ascorbate make only a small impact, if any, on CN II and CN I expression.		#*You can see that in 2D culture, the additions of proline and ascorbate make only a small impact, if any, on CN II and CN I expression.
	#Finally, look briefly at your melt curves. As we discussed in lecture, the samples should each exhibit a single peak that can be attributed to the melting of the desired product. There may also be a small primer-dimer peak, about 10% of the main peak intensity. Do any of your samples have a substantial amount of non-specific (most likely primer-dimer) product?		#Finally, look briefly at your melt curves. As we discussed in lecture, the samples should each exhibit a single peak that can be attributed to the melting of the desired product. There may also be a small primer-dimer peak, about 10% of the main peak intensity. Do any of your samples have a substantial amount of non-specific (most likely primer-dimer) product?
-	#*T/R: ~~Well E3 gave a primer dimer peak, while wells E4, E7-8, E11-2, D6, and D12 were flat. Well C10 is offset to a slightly lower Tm. Wells D5, C6, and C8 are shifted to a slightly higher Tm.~~	+	#*T/R:
-	#*W/F: ~~Well A7 (CN I) had a shifted Tm (to a lower value). Well C3 had an odd-shaped curve indicating primer-dimer and/or other non-specific products. Wells as listed above gave flat curves.~~	+	#*W/F:
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AgiStachowiak: /* Part 2: Proteoglycan assay */

2013-05-03T15:52:04Z

Part 2: Proteoglycan assay

←Older revision		Revision as of 15:52, 3 May 2013
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	The colorimetric response to the formation of DMMB-GAG complexes is short-lived, and thus the absorbances must be measured within 5 minutes of combining the dye and samples. Tell the teaching faculty when you plan to do the assay. With their go ahead, add the standards and samples to the plate. Then, you will go upstairs to the BPEC facility with an instructor and add the dye then and there.		The colorimetric response to the formation of DMMB-GAG complexes is short-lived, and thus the absorbances must be measured within 5 minutes of combining the dye and samples. Tell the teaching faculty when you plan to do the assay. With their go ahead, add the standards and samples to the plate. Then, you will go upstairs to the BPEC facility with an instructor and add the dye then and there.

-	#You will be given chondroitin sulfate (CS) at 200 μg/mL. Prepare ~~100 μL~~ doubling dilutions of this GAG in water that contains 0.15 % alginate.	+	#You will be given chondroitin sulfate (CS) at 200 μg/mL. Prepare doubling dilutions of this GAG in water that contains 0.15 % alginate such that you will have 100 μL of each left over for distribution.
	#*Alginate is included in order to account for any slight background response that it may still have at pH 1.5.		#*Alginate is included in order to account for any slight background response that it may still have at pH 1.5.
	#Add 40 μL duplicate samples of CS to columns 1 and 2 of your plate, in all eight rows A-H.		#Add 40 μL duplicate samples of CS to columns 1 and 2 of your plate, in all eight rows A-H.

AgiStachowiak: /* Part 2: Proteoglycan assay */

2013-05-03T15:49:52Z

Part 2: Proteoglycan assay

←Older revision		Revision as of 15:49, 3 May 2013
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	===Part 2: Proteoglycan assay===		===Part 2: Proteoglycan assay===

-	The colorimetric response to the formation of DMMB-GAG complexes is short-lived, and thus the absorbances must be measured within 5 minutes of combining the dye and samples. Tell the teaching faculty when you plan to do the assay. With their go ahead, add the standards and samples to the plate. Then, you will upstairs to the BPEC facility with an instructor and add the dye there.	+	The colorimetric response to the formation of DMMB-GAG complexes is short-lived, and thus the absorbances must be measured within 5 minutes of combining the dye and samples. Tell the teaching faculty when you plan to do the assay. With their go ahead, add the standards and samples to the plate. Then, you will go upstairs to the BPEC facility with an instructor and add the dye then and there.

	#You will be given chondroitin sulfate (CS) at 200 μg/mL. Prepare 100 μL doubling dilutions of this GAG in water that contains 0.15 % alginate.		#You will be given chondroitin sulfate (CS) at 200 μg/mL. Prepare 100 μL doubling dilutions of this GAG in water that contains 0.15 % alginate.