Transcriptomics Flashcards
What is transcriptomics?
Analysis of RNA abundance/expression.
Why can it be relevant to analyze levels of RNA expression?
Differences in phenotype can be be due to differences in gene expression, even though the DNA is identical. By analyzing differences in RNA levels, we can identify the causes for the phenotypic differences.
We can also locate ncRNAs and find their functions.
Why don’t we use just proteomics to analyze gene expression?
Many differences in expression is seen already at RNA level, and its also easier to detect RNA (mainly since the molecule is very homogenous compared to proteins). Transcriptomics is less biased than proteomics, and also cheaper.
Why is transcriptomics less biased than proteomics?
Because RNA is a very homogenous molecule compared to proteins. Therefore, when detecting/measuring levels of proteins, the tools need to be modified specifically after a particular type of protein, since all are very different. It’s difficult to measure the global level of proteins.
What are the most common methods for RNA quantification?
PCR based- RT-qPCR: low throughput
Hybridization based:
Northern blot: low throughput
Microarray: high throughput
Sequencing based:
RNA-Seq: high throughput
In what ways are RNA-Seq advantageous to Microarrays?
Analysis of RNA with microarrays is limited to a predefined set of RNA probes that are present on the chip. If you don’t know the sequence of the gene you’re investigating, then it won’t be detectable. RNA-seq can be used to detect RNA levels of any gene.
How can transcriptomics be used to find isoforms of a gene? What sequencing method is the best fit for this?
RNA-seq can be used to examine the alternative splicing one or multiple genes, since mRNA transcripts are transcribed into cDNA by RT. The transcript abundance can be compared between different samples to find isoforms of the same gene, for ex. between different conditions. Nanopore is a good method for this, since it generates long reads.
How can RNA-seq be used to detect RNA editing sites?
One common example of RNA editing is the ADAR enzyme converting an adenosine to an inosine. This will be read as a guanosine in the sequencing, and the basepairing will show up as G-C. By comparing the sequenced RNA to a reference, A–>G mutations can indicate an editing event.
What is Cross-linking immunoprecipitation (CLIP)?
The purpose of CLIP is to determine RNA-protein interactions, to where in an RNA specific proteins bind.
Cells are exposed to UV light to create covalent bonds between protein and RNA. Then the cells are lysed, and the protein of interest is isolated via immunoprecipitation (a tagged antibody is added, binds to specific protein). RNA is isolated and washed, then cDNA is generated using RT-PCR, followed by sequencing.
