Summary
Sequencing of cDNA from RNA to quantify gene expression and discover transcripts across the genome.
In depth
RNA is converted to cDNA, sequenced, and reads mapped to a reference to count transcripts. It finds splice variants, novel genes and differential expression, with a wide dynamic range. Normalisation (TPM) and statistics (DESeq2) are essential.
Poly-A selection or rRNA depletion enriches mRNA. Reads are counted per gene or transcript; replicate samples allow statistical testing. Spatial transcriptomics adds tissue location.
Example: RNA-seq of infected cells shows which host genes respond.
RNA-seq measures all RNA in a sample. RNA is extracted, often enriched for mRNA, converted to cDNA, fragmented, sequenced, and reads are aligned to a genome or transcriptome.
Counts of reads per gene are normalised (for example TPM) and compared between conditions to find differentially expressed genes. It also detects splice variants, fusion genes, non-coding RNAs and novel transcripts, with no need for prior probes. Replicates and proper statistics are needed.
Example: comparing tumour and normal tissue reveals pathways switched on in cancer.
Key idea: RNA levels give a snapshot of cell state but do not always match protein levels.
Frequently asked questions
What is RNA-Seq?
Sequencing of cDNA from RNA to quantify gene expression and discover transcripts across the genome.
Which area of biology covers RNA-Seq, and how advanced is it?
RNA-Seq is one of the 56 topics in Biotechnology & Genomics. It is rated Advanced, so it suits learners who want specialist depth. Related topics in this area include DNA Microarrays, Functional Genomics, Bioinformatics.
Can you give an example of RNA-Seq?
Comparing tumour with normal tissue reveals pathways.
What should I learn before RNA-Seq?
Work through the Beginner topics in Biotechnology & Genomics first, such as Biotechnology, Genetic Engineering, Selective Breeding, then return to RNA-Seq. Advanced topics assume the Intermediate vocabulary.
What topics are related to RNA-Seq?
The closest topics to RNA-Seq are DNA Microarrays, Functional Genomics, Bioinformatics, Single-Cell Sequencing. Comparing them side by side is a good way to see how the ideas connect.
How can I check that I understand RNA-Seq?
Read the summary and the in-depth section, then use the quick check on the interactive lesson for RNA-Seq. To practise the whole of Biotechnology & Genomics, use the Biotechnology & Genomics quiz.
How is RNA-Seq different from DNA Microarrays?
RNA-Seq: Sequencing of cDNA from RNA to quantify gene expression and discover transcripts across the genome. DNA Microarrays: Slides carrying thousands of probes that measure expression of many genes at once. Read both side by side and note what each one explains.
How should I study RNA-Seq step by step?
First, read the summary and write the definition in your own words. Second, read the in-depth section and note any example. Third, compare it with DNA Microarrays and explain the difference aloud. Finally, take the Biotechnology & Genomics quiz and come back to this page for any question you miss.