Biomedical Science Experiment Protocol -- 2
Budget: $10 – $30 USD
There are two parts. Part 1 is to produce a protocol using relevant commercial kits to address the hypothesis:
“Compound X a new anti-TGF beta targeting compound prevents TGF-beta-induced decreases in E-cadherin mRNA expression in PC-3 prostate cancers cells in-vitro using sybr green based quantitative PCR”
To write the protocol you will need to perform a literature search to identify relevant commercial kits that you will base your protocol around. Word count 1300 words maximum, excluding references in the reference list (Harvard style), figures / tables and their legends (which should be concise). Your protocol will consist of three sections:
Section 1. Using Sigma GenElute™ Total RNA Purification Kit. to isolate mRNA from the following PC3 cell cultures.
. Untreated PC3 Cells (6 replicates) - "basal control”
PC3 cells treated with TGF-beta (6 replicates) - e-cadherin expression should be suppressed - "positive control”
. PC3 cells treated with TGF-beta and compound X (6 replicates) - "experimental group”
. PC3 cells treated with compound X (6 replicates) - "experimental group”
Section 2. Reverse transcription of isolated mRNA into cDNA using Sigma ReadyScript® cDNA Synthesis Mix.
Section 3. Quantitative PCR to establish copy number for your target gene (e-cadherin) and a housekeeping gene (beta actin) using SYBR green based Quantitative PCR. This is for the above groups and two sets of standards of known copy number; one for e-cadherin and another for Beta actin (to create two standard curves to enable quantification).
Part 2 Is the analysis and description of results. The word count should be 300 words maximum including references, figure and table legends. This will require the abstraction and manipulation of the data, evaluation and synthesis of the data, backed up by appropriate statistical analyses to produce a written description (not discussion i.e., do not include any biological reason for the changes or if the assay is valid), supported with appropriate tables and a maximum of one figure (a bar chart). There are four groups and one quantitative variable. You can use any statistical/graphing programme e.g., Minitab, SPSS, Excel to perform this analysis. Remember when describing the outcome of your analysis you need to present it in a clear and easily interpretable manner with the statistics annotated on the figure and this annotation described in the figure legend. Look at the example in figure 1 and other figures published in journals to see how statistical analysis is incorporated into figures. Don’t present the “raw” outcomes from Minitab or describe the statistical process, describe your interpretation of the analysed data to the reader.
Another common mistake is to over analyse the data; concentrate on what you consider to be the key variables/groups. Having established this, you need to concentrate your description around changes in these variables and groups. If you are uncertain if a particular descriptive analysis is adding anything to the analysis omit it. Extra descriptive statistics can be incorporated into a table. The description could include but not be limited to the following median, standard error of the mean, ranges. Don’t present raw data.
You should use a single figure containing a bar chart presenting mean changes in the groups’ target mRNA expression with standard deviations. The figure should have labelled axes with an appropriate legend and should clearly present the data and key statistical differences. An appropriate table containing additional descriptive data can be used to add further depth of understanding.
Data used in Part 2 can be found in the attached files.
“Compound X a new anti-TGF beta targeting compound prevents TGF-beta-induced decreases in E-cadherin mRNA expression in PC-3 prostate cancers cells in-vitro using sybr green based quantitative PCR”
To write the protocol you will need to perform a literature search to identify relevant commercial kits that you will base your protocol around. Word count 1300 words maximum, excluding references in the reference list (Harvard style), figures / tables and their legends (which should be concise). Your protocol will consist of three sections:
Section 1. Using Sigma GenElute™ Total RNA Purification Kit. to isolate mRNA from the following PC3 cell cultures.
. Untreated PC3 Cells (6 replicates) - "basal control”
PC3 cells treated with TGF-beta (6 replicates) - e-cadherin expression should be suppressed - "positive control”
. PC3 cells treated with TGF-beta and compound X (6 replicates) - "experimental group”
. PC3 cells treated with compound X (6 replicates) - "experimental group”
Section 2. Reverse transcription of isolated mRNA into cDNA using Sigma ReadyScript® cDNA Synthesis Mix.
Section 3. Quantitative PCR to establish copy number for your target gene (e-cadherin) and a housekeeping gene (beta actin) using SYBR green based Quantitative PCR. This is for the above groups and two sets of standards of known copy number; one for e-cadherin and another for Beta actin (to create two standard curves to enable quantification).
Part 2 Is the analysis and description of results. The word count should be 300 words maximum including references, figure and table legends. This will require the abstraction and manipulation of the data, evaluation and synthesis of the data, backed up by appropriate statistical analyses to produce a written description (not discussion i.e., do not include any biological reason for the changes or if the assay is valid), supported with appropriate tables and a maximum of one figure (a bar chart). There are four groups and one quantitative variable. You can use any statistical/graphing programme e.g., Minitab, SPSS, Excel to perform this analysis. Remember when describing the outcome of your analysis you need to present it in a clear and easily interpretable manner with the statistics annotated on the figure and this annotation described in the figure legend. Look at the example in figure 1 and other figures published in journals to see how statistical analysis is incorporated into figures. Don’t present the “raw” outcomes from Minitab or describe the statistical process, describe your interpretation of the analysed data to the reader.
Another common mistake is to over analyse the data; concentrate on what you consider to be the key variables/groups. Having established this, you need to concentrate your description around changes in these variables and groups. If you are uncertain if a particular descriptive analysis is adding anything to the analysis omit it. Extra descriptive statistics can be incorporated into a table. The description could include but not be limited to the following median, standard error of the mean, ranges. Don’t present raw data.
You should use a single figure containing a bar chart presenting mean changes in the groups’ target mRNA expression with standard deviations. The figure should have labelled axes with an appropriate legend and should clearly present the data and key statistical differences. An appropriate table containing additional descriptive data can be used to add further depth of understanding.
Data used in Part 2 can be found in the attached files.