Stem-Loop Primers vs Two-Tailed Primers


Small RNAs, such as siRNA, miRNA, snoRNA, and others ranging from approximately 18 to 26 nucleotides, cannot be amplified using conventional PCR primers, as a single primer matches their entire length. Various techniques are used to elongate short RNA molecules to address this limitation, enabling them to be amplified during qPCR or dPCR. However, a critical consideration across these techniques is maintaining specificity—ensuring that only the intended target is amplified—and sensitivity, allowing the detection of even low-abundance targets such as therapeutic siRNA in tissues or biofluids.


stem-loop primer used in a qPCR instrument

The stem-loop primer has a 6 bp-long region that binds complementarily to the last six nucleotides at the 3’ end of the target short RNA. In contrast, the two-tailed primer has two sites, called hemiprobes, that bind complementary to each end of the short RNA. Both hemiprobes need to hybridize with the target, thereby increasing specificity.

In the stem-loop approach, only one primer in the qPCR/dPCR is specific to the target, while the two-tailed primer consists of a target-specific forward and reverse primer. This additional specificity further enhances the accuracy of the method.

RNAi using stem-loop and two-tail

Download our guide on how to setup a PCR-workflow for short targets.

For the PCR-based bioanalytical methods for RNAi therapeutics.


Assay design

The two-site interaction with the two-tailed primer allows for greater primer design flexibility than the stem-loop primer, which binds exclusively to the 3′ end of the short RNA without flexibility. The base pairs in that region determine the GC content, melting temperature, and other parameters of the PCR reaction, limiting adaptability. A two-tailed primer design can be optimized for specific PCR conditions and validation requirements.

While the stem-loop primer is easier to design than the two-tailed primer, at TATAA, we have developed hundreds of assays using the two-tailed approach. Contact us, and we can assist.

How the stem-loop primer works:

  1. The stem-loop primer binds to the 3′ end of the short RNA.
  2. The stem-loop primer serves as a primer for the reverse transcriptase during cDNA synthesis (stem-loop RT).
  3. The resulting cDNA contains both the original short RNA sequence and the primer sequence, now extended enough to be quantified using a conventional dPCR/qPCR approach. The forward primer is complementary to the short RNA, and the reverse primer is complementary to the elongated primer sequence.

How the Two-Tail primer works:

  1. The two-tail primer is designed with two hemiprobe sequences that bind to the short RNA. Each hemiprobe alone cannot bind, so the two-site approach increases specificity.
  2. The reverse transcriptase extends from one end, creating a cDNA.
  3. The resulting cDNA, containing both the two-tailed primer and the short RNA, can now be quantified using conventional qPCR/dPCR. In contrast to the stem-loop primer, both the forward and reverse primers complement the short RNA of interest.


Watch the Two-Tail video here

Modifications of the two-tail approach can be used to detect ASO or other short DNA. Contact us for more information on that. 

TATAA Biocenter invented the Two-Tailed RT-PCR technique, and we have used it for various customer projects involving short RNA or DNA, such as siRNA, miRNA, and ASO. Additionally, we have employed the Two-Tailed method for applications requiring high specificity, including detecting single-base differences.

TATAA Biocenter specializes in qPCR, dPCR, and NGS for CGT and advanced therapy drug development. We conduct qPCR and dPCR assays by GLP accreditation, GCLP compliance, and ISO/IEC 17025 standards. Our services include developing, optimizing, qualification, and validating assays tailored to each project and target.