Despite RNA-Seq being the capstone technology for gene expression profiling, qPCR remains the go-to technique for validation. To this day, qPCR validation of microarray data is commonplace, and many see it as necessary, given that microarrays involve hybridization to a glass slide. Furthermore, the dynamic range of microarrays has long been known to be constrained compared to qPCR. For those with RNA-Seq data, being asked for qPCR validation is often met with surprise. Let’s take a look at situations when qPCR validation of RNA-Seq data is appropriate and inappropriate.
When qPCR validation is appropriate:
1) When a second method is necessary to confirm an observation. In other words, everyone involved might not completely believe the observation until it’s confirmed using a different approach. Think of this as the “journal reviewer” mindset. Academic investigators often perform qPCR validation of their RNA-Seq results to ensure their manuscripts get published in the journal of their choice. The reviewers ultimately want to see the same results obtained using different techniques.
2) When your RNA-Seq data is based on a small number of biological replicates. Let’s call this the “cost savings” mindset. Some investigators use few or no replicates in their RNA-Seq studies in order to keep costs down. The number of replicates can be so low that proper statistical tests cannot be applied. In these cases, using qPCR on more samples, and focusing on a few interesting targets, is a great method for validating the RNA-Seq results and building out the study.
In both cases, it’s important to note that the qPCR experimental workflow is much simpler than the RNA-Seq workflow. With that simplicity, comes less risk for introducing bias. Finally, qPCR is a mature technology that has withstood the test of time, and for that reason, remains a popular method of validating RNA-Seq data. You can rest assured that qPCR will ultimately increase your confidence in the RNA-Seq data.
When qPCR validation is inappropriate:
1) When the RNA-Seq data is only a small part of the story. Let’s call this the “primary screen” mindset. If you’re using the RNA-Seq results to generate new hypotheses that will be exhaustively tested at a more focused level, then qPCR validation is not always necessary. This is particularly relevant to protein-level approaches. If you jump to protein-level approaches after being informed by the RNA-Seq data, then it’s reasonable to assume that qPCR studies aren’t worth the time and resources.
2) When you plan on doing more RNA-Seq. Many believe that suitable validation of RNA-Seq data is generating more RNA-Seq data on a new, larger set of samples. The rationale here is that if you get the same results in two separate data sets, then the results are likely true no matter what technology is used.
Conclusion: Validating RNA-Seq results with qPCR
If you plan to validate your RNA-Seq results using qPCR, then we highly recommend using a different set of samples with proper biological replication. Performing qPCR on the same set of RNA samples used for RNA-Seq is a good control for technology – hopefully, you get the same result using 2 different techniques. Performing qPCR on a new set of RNA samples gives you more power – you’ve validated the technology, the results, and the underlying biological response. Now that’s a win-win situation!