Base Pair Breakdown: Simplifying 3 methods of analyzing nucleic acid data
Differentiating between genome and transcriptome analysis techniques. When to use RNA-Seq vs RT-qPCR vs online databases such as Ensembl.
RNA-Seq
Lab procedure to analyze an entire, unknown transcriptome for differential gene expression and processing analysis
RT-qPCR
Lab procedure to detect and quantify known, targeted DNA sequences
Ensembl
Genome browser to compare known orthologs, or the same gene across multiple species
Key Terms
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Genome: All DNA in the nucleus and mitochondria of a cell
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Transcriptome: All RNA in the nucleus and mitochondria of a cell. A direct product of genome expression.
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Differential Gene Expression: Comparison of expression levels of a certain gene between two experimental conditions, often as a result of epigenetic changes.
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Ortholog: The same gene across different species. Gene function is conserved but sequence may not be.
Introduction
It is often intimidating to receive a data file, be instructed to explore genes on the internet, or start an experiment without fully knowing the biology background. If you know how to use the tools correctly and what type of data you are dealing with, so much analysis can be done with information already online. Here are three different methods of genome and transcriptome level analysis so that you can find the technique that is best suited for the information you have, and so that you do not feel lost when reading papers that refer to these methods!
RNA-Seq
Purposes: Differential gene expression analysis, transcriptional profiling, information about alternative splicing
Input Material: Transcriptome (total cell content of mRNA, rRNA, and tRNA)
Use When: You have a novel transcript that is not on online databases (i.e. Cuvier's beaked whale), you are running experiments on cells and want to analyze transcriptomes under specific conditions
Example of use: Expose whale and human cells to normoxia and hypoxia and use RNA-Seq to determine which genes are more highly expressed or have decreased expression
RT-qPCR
aka Reverse Transcription quantitative real time Polymerase Chain Reaction
Purposes: Quantify expression of a known gene
Input Material: Total RNA and single stranded DNA primers to amplify cDNA.
Use When: You have a specific gene of interest with a known sequence and you want to quantify that target DNA sequence in reference to gene copy number, rather than differential expression
Example of use: I have a gene PGC1a and I want to know if there are more copies in whales versus human cells. I can design primers for the gene in whales and the gene in humans and run RT-qPCR.
Ensembl
Purposes: Compare annotated genomes between species in pre-loaded database with the ability to upload your own genomes for comparison.
Input Material: Pre-loaded DNA sequences or genome sequences from outside databases for species of choice.
Use When: You want to compare gene sequences between species. This will not tell you about the RNA level of species not in their database. Used in conjunction with NCBI Blast.
Example of use: I want to design primers for PCR, but my genome of a whale is not annotated. I align the known sequence from the human genome of the gene of interest from Ensembl to the genome of the whale from an outside database using Blastn.
An example using NCBI online genome browser to design oligonucleotides for CRISPR