Development of a 3′:5′ digital PCR assay to determine horse mRNA integrity.

The research article presents the development of a 3′:5′ digital PCR assay which enables more reliable and rapid measurements of RNA integrity in horse samples. This method, unlike its predecessor, also allows for a direct evaluation of the quantity of messenger RNA (mRNA).

Development and Importance of the Assay

• The scientists focused on developing a 3′:5′ digital PCR (dPCR) assay, primarily aiming to improve the accuracy of measurements of RNA integrity.
• RNA integrity refers to the notion that RNA molecules in a sample are complete and functional.
• Measuring RNA integrity is crucial as its damage or fragmentation can drastically affect subsequent analyses, including gene expression studies.
• The reliability of gene expression data, which is critical in many fields of biological research including studies on diseases, depends significantly on the integrity of the RNA used.

Limitations of Previous Methods

• The previously employed method, known as reverse transcription quantitative PCR (RT-qPCR) 3′:5′ assay, had its limitations.
• It required the use of standard curves, which could introduce variations and inaccuracies in the measurements. Standard curves are graphical representations that establish the relationship between two quantities and are used to interpret the collected data.
• This RT-qPCR method also had a potential issue with PCR inhibitors, which can interfere with the reaction by binding to the components of the PCR or changing the conditions needed for the PCR to work.
• Especially when working with small samples, these factors make the method cumbersome and the results increasingly unreliable.

Benefits of the Developed 3′:5′ dPCR Assay

• The new 3′:5′ dPCR assay seems to provide a more direct determination of messenger RNA (mRNA) integrity, thus increasing the precision of the results.
• Not only does it measure the mRNA integrity, but it also provides information about the quantity of mRNA present in the sample, which is an added advantage over the RT-qPCR based 3′:5′ assay.
• The scientists suggest that this newly created dPCR assay could act as a rapid, uncomplicated alternative for assessing RNA integrity in equine samples.