Gene Expression Analysis: RNA-Seq or Real-Time PCR?

Although the underlying principles behind RNA-seq and real-time PCR are different, both can provide information about the amount (absolute or relative) of mRNA isolated from any sample type. If you’re new to research or starting gene expression analysis for the first time, you might be wondering: if both techniques can do the job, why choose one over the other? In this post, we’ll help you answer that question!

RNA-seq uses next-generation sequencing (NGS) technology to provide a snapshot of the quantity and identity of RNA molecules in a sample at a specific time under conditions of interest. To specifically analyze mRNA, an mRNA enrichment step can be added to the workflow. The mRNA in the enriched samples is then reverse transcribed into complementary DNA (cDNA), which is subsequently converted into a sequencing library. This library is sequenced on an instrument, and the output is processed into quantitative lists of transcripts present in each sample. The output of an RNA-seq experiment includes most or all of the mRNAs in the sample(s) at the time of collection. In other words, RNA-seq is a broad, catch-all technique.

Real-time PCR, on the other hand, is a more targeted approach to gene expression analysis. Total RNA is reverse transcribed into cDNA, which is amplified using target-specific primers in a quantitative PCR reaction. Fluorescent DNA-binding probes (target-specific) or fluorescent dsDNA-binding dyes (non-specific) are used in the PCR reaction, and the level of fluorescence emitted during amplification reflects the amount of target present in the sample. Because real-time PCR requires target-specific primers and/or probes for each gene of interest, it is not a catch-all technique. However, with a good set of target-specific probes, it is possible to multiplex—analyzing several targets in the same sample simultaneously.

It’s not accurate to say one technique is inherently better than the other. Both offer sensitive and reliable transcript quantification, but depending on your research goals, one might suit your needs better. Let’s explore some key factors to consider when deciding between the two.

1. How many genes are you looking at?

If you’re interested in studying the entire transcriptome to see how it responds to a particular treatment or condition, RNA-seq is the way to go. It provides a comprehensive view of how all genes in your organism are upregulated, downregulated, or unchanged in response to your chosen experimental conditions.

This transcriptome-wide approach is especially valuable in the early stages of a project, when you might not yet know which genes to focus on. Examples include:

• Identifying potential disease-associated genes by comparing the transcriptomes of healthy and diseased tissues
• Investigating the mechanism of action of a compound by examining how it impacts gene expression
• Studying the overall transcriptional response to environmental compounds like pesticides or fungicides
• Characterizing a transcription factor by knocking it out/down and analyzing which genes are regulated by it

If, however, you already know which genes are of interest, RNA-seq might be unnecessary. With well-designed primers and a reliable real-time PCR setup, you can efficiently detect and quantify your target genes. Unless you’re working with a large number of genes, real-time PCR is also likely to be more cost-effective than RNA-seq.

2. Are you interested in studying gene regulation?

If your lab typically studies a specific group of genes using real-time PCR but now wants to understand how those genes are regulated, RNA-seq could be a great starting point. For example, you could analyze the transcriptional changes in all regulatory genes and transcription factors in response to activating your target pathways.

Additionally, RNA-seq is ideal for simultaneously studying gene expression and regulation. By skipping the mRNA enrichment step before cDNA synthesis, RNA-seq can capture all RNA molecules present, including mRNAs, tRNAs, small non-coding RNAs, and others, offering insights into both coding and non-coding RNA regulation.

3. Want to go into discovery mode?

Real-time PCR requires prior knowledge of the genes you want to study because it depends on target-specific primers (and probes unless using non-specific dsDNA-binding dyes like SYBR Green). This makes it unsuitable for discovering new genes, alternative transcripts, or unannotated variants.

In contrast, RNA-seq doesn’t require prior sequence knowledge. It’s the perfect tool for hypothesis-free, large-scale gene discovery. Use RNA-seq if you want to identify new genes, transcripts, or variants in your system of interest.