Difference between revisions of "20.109(S13):Preparing cells for analysis (Day4)"
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− | <font color=red> | + | <font color=red>Done. Light edits completed Monday morning.</font color> |
==Introduction== | ==Introduction== | ||
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Folks trying to engineer cartilage tissue have been in interested in this and similar questions for some time. After all, the more closely an ''in vitro'' or ''in vivo'' model construct can mimic natural tissue and promote its development, the more successful it may be for wound and disease repair. Engineering tissue thus requires an expert understanding of what the native tissue is like. Articular cartilage is a water-swollen protein network consisting of >50% collagen Type II, along with small amounts of collagen Types IX and XI. The collagen fibrils vary in diameter, cross-linking density, and orientation (random or aligned) depending on the depth of the tissue cross-section that is examined (see figure). Unlike cartilage, many other connective tissues are composed primarily of collagen Type I. | Folks trying to engineer cartilage tissue have been in interested in this and similar questions for some time. After all, the more closely an ''in vitro'' or ''in vivo'' model construct can mimic natural tissue and promote its development, the more successful it may be for wound and disease repair. Engineering tissue thus requires an expert understanding of what the native tissue is like. Articular cartilage is a water-swollen protein network consisting of >50% collagen Type II, along with small amounts of collagen Types IX and XI. The collagen fibrils vary in diameter, cross-linking density, and orientation (random or aligned) depending on the depth of the tissue cross-section that is examined (see figure). Unlike cartilage, many other connective tissues are composed primarily of collagen Type I. | ||
− | Extracellular matrix (ECM) proteins such as the collagens must be synthesized by cells. Chondrocytes readily synthesize collagen II, while fibroblasts and mesenchymal stem cells primarily synthesize collagen I. Thus, the expression and production of different collagens is one way to distinguish these cells types. To study collagen at the gene transcript level, you will break open and homogenize your cells using a lysis reagent and column (QIAshredder) and then isolate RNA using an RNeasy kit from Qiagen. The RNeasy kit includes silica gel columns, similar to the ones you used to purify DNA in Module 1, that selectively bind RNA (but not DNA) that is >200 bp long under appropriate buffer conditions. Due to size exclusion, the resultant RNA is somewhat enriched in mRNAs relative to rRNA and tRNA. To further purify for mRNA, one could use a polyT affinity column to capture the polyA tail of this RNA type, but we will not do this today. | + | Extracellular matrix (ECM) proteins such as the collagens must be synthesized by cells. Chondrocytes readily synthesize collagen II, while fibroblasts and mesenchymal stem cells primarily synthesize collagen I. Thus, the expression and production of different collagens is one way to distinguish these cells types. To study collagen at the gene transcript level, you will break open and homogenize your cells using a lysis reagent and column (QIAshredder) and then isolate RNA using an RNeasy kit from Qiagen. The RNeasy kit includes silica gel columns, similar to the ones you used to purify DNA in Module 1, that selectively bind RNA (but not DNA) that is >200 bp long under appropriate buffer conditions. Due to size exclusion, the resultant RNA is somewhat enriched in mRNAs relative to rRNA and tRNA. To further purify for mRNA, one could use a polyT affinity column to capture the polyA tail of this RNA type, but we will not do this step today. |
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− | After eluting and measuring your total RNA, you will perform a reverse transcription (RT) reaction to make cDNA from the mRNA. Next time you will amplify the gene transcripts of interest, namely those for the collagen I and collagen II alpha chains, by PCR. In previous iterations of this module, we used | + | After eluting and measuring your total RNA, you will perform a reverse transcription (RT) reaction to make cDNA from the mRNA. Next time you will amplify the gene transcripts of interest, namely those for the collagen I and collagen II alpha chains, by PCR. In previous iterations of this module, we used a 1-step RT-PCR kit, and then ran the amplified cDNA products out on an agarose gel to compare the changes in collagen II and I expression for the two culture conditions. However, as you may have noticed by now, agarose gels do not have a large dynamic range. Moreover, end-point PCR is prone to error should the conditions (e.g., amount of RNA) deviate from those used to optimize the assay. For these reasons, we now use a more sensitive method for quantifying the transcripts, called real-time-PCR or sometimes RT-PCR, confusingly! The method is also called qPCR, for quantitative PCR. |
In qPCR, the amount of DNA is measured after each cycle of PCR, in contrast to an end-point PCR assay. The DNA is detected by using a dye that fluoresces only when it binds to DNA (similar to ethidium bromide staining), or even a tagged primer that fluoresces only when it binds to the desired product. As the DNA is amplified, fluorescence is repeatedly measured and increases exponentially over time. Finally, cDNA product renaturing competes with primer annealing and the fluorescence intensity plateaus rather than growing. Comparisons between samples are done using data in the exponential regime. | In qPCR, the amount of DNA is measured after each cycle of PCR, in contrast to an end-point PCR assay. The DNA is detected by using a dye that fluoresces only when it binds to DNA (similar to ethidium bromide staining), or even a tagged primer that fluoresces only when it binds to the desired product. As the DNA is amplified, fluorescence is repeatedly measured and increases exponentially over time. Finally, cDNA product renaturing competes with primer annealing and the fluorescence intensity plateaus rather than growing. Comparisons between samples are done using data in the exponential regime. | ||
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When measuring changes in gene expression, primer design must be appropriate for a cDNA rather than genomic DNA. For example, a single primer that includes sequence from two neighbouring exons (along with a second primer that has sequence from just one exon) will amplify mRNA but not genomic DNA, which may be present as a contaminant (see also figure at right). What will happen if each primer contains sequence from only one exon? Primer design and quality control for qPCR also entail further consideration, particularly if tagged primers are used. In our case, the true efficiency of the primers needed to be measured (maximum efficiency is 2, i.e., perfectly exponential growth) to enable later quantification of transcript level changes. You will learn more about qPCR analysis in the coming week. | When measuring changes in gene expression, primer design must be appropriate for a cDNA rather than genomic DNA. For example, a single primer that includes sequence from two neighbouring exons (along with a second primer that has sequence from just one exon) will amplify mRNA but not genomic DNA, which may be present as a contaminant (see also figure at right). What will happen if each primer contains sequence from only one exon? Primer design and quality control for qPCR also entail further consideration, particularly if tagged primers are used. In our case, the true efficiency of the primers needed to be measured (maximum efficiency is 2, i.e., perfectly exponential growth) to enable later quantification of transcript level changes. You will learn more about qPCR analysis in the coming week. | ||
− | Next time (day 5) | + | Next time (day 5) we ll initiate an assay called ELISA to observe collagen at the protein (rather than transcript) level and also begin analysis of the data collected thus far. |
==Protocols== | ==Protocols== | ||
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Before (second TC cohort) or after (first TC cohort) your wet-lab work today, take some time to discuss the five research results you wrote up for homework with your lab partner, guided by the instructions below. | Before (second TC cohort) or after (first TC cohort) your wet-lab work today, take some time to discuss the five research results you wrote up for homework with your lab partner, guided by the instructions below. | ||
− | Writing a research proposal requires that you identify an interesting topic, spend lots of time learning about it, and then design some clever experiments to advance the field. It also requires that you articulate your ideas so any reader is convinced of your expertise, your creativity and the significance of your findings, should you have the opportunity to carry out the experiments | + | Writing a research proposal requires that you identify an interesting topic, spend lots of time learning about it, and then design some clever experiments to advance the field. It also requires that you articulate your ideas so any reader is convinced of your expertise, your creativity and the significance of your findings, should you have the opportunity to carry out the experiments you ve proposed. To begin you must identify your research question. This may be the hardest part and the most fun. Fortunately you started by finding a handful of topics to share with your lab partner. Today you should discuss and evaluate the topics you ve gathered. Consider them based on: |
* your interest in the topic | * your interest in the topic | ||
* the availability of good background information | * the availability of good background information | ||
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* if your proposal could be carried out in a reasonable amount of time and with non-infinite resources | * if your proposal could be carried out in a reasonable amount of time and with non-infinite resources | ||
− | It might be that not one of the topics | + | It might be that not one of the topics you ve identified is really suitable, in which case you should find some new ideas. It s also possible that through discussion with your lab partner, you ve found something new to consider. Both of these outcomes are fine but by the end of today s lab you should have settled on a general topic or two so you can begin the next step in your proposal writing, namely background reading and critical thinking about the topic. '''Check in with Thomas and get his feedback about your ideas for a few minutes before leaving today.''' |
A few ground rules that are 20.109 specific: | A few ground rules that are 20.109 specific: | ||
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*You should keep in mind that this proposal will be presented to the class, so try to limit your scope to an idea that can be convincingly presented in a twelve minute oral presentation. | *You should keep in mind that this proposal will be presented to the class, so try to limit your scope to an idea that can be convincingly presented in a twelve minute oral presentation. | ||
− | Once you and your partner have decided on a suitable research problem, | + | Once you and your partner have decided on a suitable research problem, it s time to become an expert on the topic. This will mean searching the literature, talking with people, generating some ideas and critically evaluating them. To keep track of your efforts, you should start a wiki catalog on your OpenWetWare user page. How you format the page is up to you but check out the [http://openwetware.org/wiki/Yeast_rebuild | |