A tool for design of primers for microRNA-specific quantitative RT-qPCR

Abstract

Background: MicroRNAs are small but biologically important RNA molecules. Although different methods can be used for quantification of microRNAs, quantitative PCR is regarded as the reference that is used to validate other methods. Several commercial qPCR assays are available but they often come at a high price and the sequences of the primers are not disclosed. An alternative to commercial assays is to manually design primers but this work is tedious and, hence, not practical for the design of primers for a larger number of targets.

Results: I have developed the software miRprimer for automatic design of primers for the method miR-specific RT-qPCR, which is one of the best performing microRNA qPCR methods available. The algorithm is based on an implementation of the previously published rules for manual design of miR-specific primers with the additional feature of evaluating the propensity of formation of secondary structures and primer dimers. Testing of the primers showed that 76 out of 79 primers (96%) worked for quantification of microRNAs by miR-specific RT-qPCR of mammalian RNA samples. This success rate corresponds to the success rate of manual primer design. Furthermore, primers designed by this method have been distributed to several labs and used successfully in published studies.

Conclusions: The software miRprimer is an automatic and easy method for design of functional primers for miR-specific RT-qPCR. The application is available as stand-alone software that will work on the MS Windows platform and in a developer version written in the Ruby programming language.

Figure 1

Position of primers for miR-specific RT-qPCR. The sequence of the forward primer (F primer) is identical to 12 – 18 nucleotides of the microRNA and may include a tag at the 5′-end. The reverse primer (R primer) is complementary to 3 – 8 nucleotides of the microRNA, followed by 15 T residues and a tail of varying length. The 15 T’s and the tail are identical to part of the RT primer sequence.

Figure 2

Flow chart of how miRprimer designs primers. F primer: forward primer; R primer: reverse primer; A: adenine residues; Tm: melting temperature.

Figure 3

Specific amplification of a target from a biological sample. Detection of ssc-miR-15a with specific primers in cDNA made from purified pig lung total RNA (see Additional file 3). A Amplification curves. B Melting curves. C Extrapolation of Cq as function of the log10 of the relative number of templates was a straight line (R2 = 0.99) with a slope of -3.40 (PCR efficiency = 96%) over 4 log10 dilutions of a pool of all the samples used in the experiment.

Figure 4

Low detection of microRNAs that are closely related to the specific target. Amplification plots and melting curves for amplification of specific targets (ssc-let-7a, ssc-miR-125b and mmu-miR-200b-3p) and closely related miRs with one base mismatch to the forward primer (ssc-let-7e), to the reverse primer (ssc-miR-125c) and to each of the primers (mmu-miR-200c-3p). The position of the mismatch is indicated with a box on the alignment of primers and microRNAs. Only the microRNA-specific bases of the reverse primers are shown in the alignment. The curves labeled “ntc” are non-template controls. The experiment was performed as previously described using the same primers for ssc-let-7a and ssc-miR-125b [9]. QPCR of mmu-miR-200c-3p was done with the Brilliant III Ultra-Fast QPCR Master Mix (Agilent, USA). The sequence of the primers can be found in Additional file 5 (ssc-miR-125b is identical to mmu-miR-125b).