BACKGROUND: Single-stranded RNA has the potential to form secondary structures that may result in intrastrand misalignment of repeats and may be responsible for DNA mutation. Two amplicons obtained from amplification of hepatitis E virus (HEV) gene by reverse transcription and nested polymerase chain reaction (RT-nPCR) were of unexpected size and had the same misalignment. They did not contain the target region between the internal priming sites but contained two fragments flanking the target region joined by a 12-base sequence instead. OBJECTIVES: To determine whether the unexpected amplicons obtained were due to secondary structures present in the HEV RNA. STUDY DESIGN: HEV RNA sequences were obtained from the GenBank database and the software DNASIS was used to predict the presence of secondary structures within the amplification target regions. The free energy barriers of the secondary structures, which indicate their stability, were also calculated. Conventional RT-nPCR protocol was subsequently modified to eliminate RNA secondary structures. RESULTS: An extensive stem-loop structure was predicted to exist between the two internal priming sites of the HEV RNA by the DNASIS software. Its free energy barrier was found to be significant and might have resulted in the deletion of the target region located between the internal priming sites. Increased temperature and addition of dimethyl sulphoxide (DMSO) in the reverse transcription step gave the expected amplicon after the nested polymerase chain reaction. CONCLUSION: Spontaneous secondary structure formation can influence the outcome of RNA gene amplification and should be considered an important factor when designing primers and adopting protocols for RNA gene amplification.