This webpage was produced as an assignment for Genetics 564, an undergraduate capstone course at the University of Wisconsin-Madison.
What is transcriptomics?
Transcriptomics is the study of the transcriptome, the complete set of transcripts (mRNA, tRNA, rRNA, etc.) and their quantity for a specific developmental stage or physiologic condition. This is usually studied in one of two ways, microarrays or RNA-seq. Microarrays are cheaper and often used for a general overview, while RNA is more expensive but comprehensive. Transcriptomic techniques are often used to study gene expression.
How to Perform RNA-seq
1. RNA purification: Solid tissue is dissociated (sometimes to a single cell) and desired types of RNA are extracted and isolated. It is important to confirm that you have the proper size of RNA at this point.
2. Library Prep (cDNA + adapters): The polyAAA tail is used to amplify mRNA then reverse transcription is used to generate cDNA by RNA second strand synthesis. This generates the sequencing library.
3. Sequencing: The short sequence reads aligned to give the sequence.
4. Analysis: Counts of replicates for each gene are taken and normalized for sequencing depth and gene length. Heat maps are often used to represent the results, as given below for "Tsc2 tumor suppressor gene mutant kidney response to genotoxic and non-genotoxic carcinogens" over time in Rattus norvegicus [1].
1. RNA purification: Solid tissue is dissociated (sometimes to a single cell) and desired types of RNA are extracted and isolated. It is important to confirm that you have the proper size of RNA at this point.
2. Library Prep (cDNA + adapters): The polyAAA tail is used to amplify mRNA then reverse transcription is used to generate cDNA by RNA second strand synthesis. This generates the sequencing library.
3. Sequencing: The short sequence reads aligned to give the sequence.
4. Analysis: Counts of replicates for each gene are taken and normalized for sequencing depth and gene length. Heat maps are often used to represent the results, as given below for "Tsc2 tumor suppressor gene mutant kidney response to genotoxic and non-genotoxic carcinogens" over time in Rattus norvegicus [1].
Fig 1. GEO data set cluster analysis "Tsc2 tumor suppressor gene mutant kidney response to genotoxic and non-genotoxic carcinogens: time course [Rattus norvegicus]"
This zoomed-in view of the heat map allows viewing of Tsc2 expression in relation to genes that show similar expression patterns under the conditions given at the top.
Discussion
Study of the transcriptome is very useful in elucidating differential gene expression. As seen above, it may be used to discover genes that may play a role in related processes or to see if one gene is expressed differently under different conditions. This may be useful in looking for the difference underlying renal cell carcinoma formation versus benign hamartomas in TSC patients. By comparing gene expression among organs or patients with/without the carcinoma, important differences may be discovered.
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References
Stemmer, K., Ellinger-Ziegelbauer, H., Ahr, H. J., & Dietrich, D. R. (2007). Carcinogen-specific gene expression profiles in short-term treated Eker and wild-type rats indicative of pathways involved in renal tumorigenesis. Cancer research, 67(9), 4052-4068.
Image References
Header: https://microarray.org/sfgf/img/oligoArray.png
Fig 1: https://www.ncbi.nlm.nih.gov/geo/gds/analyze/analyze.cgi?ID=GDS2901
Fig 2: https://www.ncbi.nlm.nih.gov/geo/tools/profileGraph.cgi?ID=GDS4296:215735_s_at
Stemmer, K., Ellinger-Ziegelbauer, H., Ahr, H. J., & Dietrich, D. R. (2007). Carcinogen-specific gene expression profiles in short-term treated Eker and wild-type rats indicative of pathways involved in renal tumorigenesis. Cancer research, 67(9), 4052-4068.
Image References
Header: https://microarray.org/sfgf/img/oligoArray.png
Fig 1: https://www.ncbi.nlm.nih.gov/geo/gds/analyze/analyze.cgi?ID=GDS2901
Fig 2: https://www.ncbi.nlm.nih.gov/geo/tools/profileGraph.cgi?ID=GDS4296:215735_s_at