From Revolution to Evolution: Stem-loop Real-time PCR

Published September 27, 2016

Kary Mullis invented polymerase chain reaction (PCR) in 1985 creating a revolution in molecular biology techniques. But it hasn’t stopped there. PCR has greatly evolved over the years. Today, we stand at a point, where we can clone micro RNAs (miRNAs) in real time!

Due to miRNA size (about 18-21 nucleotides long) and varied expression levels, detection and quantification of miRNAs is a technically challenging task.

However, the development of the stem-loop real-time PCR technique by Chen et al.¹, provides “…a novel real-time quantification method for accurate and sensitive detection of miRNAs and other small RNAs”.

Advantages to Stem-loop Real-time PCR

Overall, the stem-loop real-time PCR technique provides the following advantages:

The technique is highly specific for miRNAs, and nonspecific genomic DNA is not amplified.
The technique is specific to mature miRNAs and not their precursors, even if both are present in equal concentrations.
You can use whole cell lysates as well as total purified RNA.
Finally, you can start with a total RNA concentration as low as 20 picograms to quantify miRNAs.

The simplified scheme for stem-loop real-time PCR includes two very familiar steps: Reverse transcription (RT) and real-time PCR. But what makes it work with miRNAs is a stem-loop primer.

The Key: The Stem-loop Primer

The stem-loop primer is the main ingredient that makes miRNA amplification feasible. These RT primers are highly specific because of the way they are designed.

The stem-loop primer consists of a constant region that forms a stem loop and a variable six- nucleotide extension. The stem-loop portion of the primer extends the ~22 nucleotide miRNA to more than ~60 nucleotides to allow for traditional PCR in subsequent steps. The six-nucleotide extension is the reverse complement of the last 6 nucleotides on the 3’ end of the miRNA of interest and provides specificity. For a great picture of a stem-loop primer and the details of how it works, check out the original article.

The success of stem-loop primers over linear primers could possibly be attributed to the stacked bases in a stem-loop, which provide thermal stability and spatial constraint that minimize errors of primer binding.

For specific information on how to design your own stem-loop primers, you can visit miRNA Design tool.

Reverse Transcription and Real-time PCR

For amplification, the miRNA is reverse transcribed using the stem-loop primer. Next, an miRNA specific forward primer, a universal reverse primer (specific for the stem-loop portion of the primer) and a dual-labeled TaqMan probe is added to the reaction to initiate a typical real-time PCR.

Troubleshooting Stem-loop Real-time PCR

You should consider several factors in optimizing your protocol.

The stem-loop primer needs to be properly folded. If it is unfolded, non-specific transcription may occur. Refolding of the primer into a stable structure takes place overnight in a regular thermocycler.
Beware of rare miRNAs: An miRNA of interest that’s not abundantly expressed can potentially generate off-targets and nonspecific quantitation. However, a combination of specific forward primer-probe pairs should minimize this noise.
Use proper controls: Make sure you include negative and positive controls. Test your protocol using synthetic RNA standards. It will help you dissect the reason for any nonspecific amplification/
Be sure that you’re using the proper equipment (i.e., PCR hood, filter pipette tips, etc.).
You need to know the precise sequence of the mature miRNA target, as this will determine the 3’ extension sequence of your stem- loop primer.

Stem-loop RT-PCR is a cost effective, convenient and consistent technique to quantify and clone miRNAs, as well as other small RNA. For once, cloning miRNAs won’t throw you for a loop!

Did you like this article? Make sure you read other PCR specific article, like ones to help you prevent PCR contamination.