Small interfering RNA (siRNA) are short oligonucleotides, which effectively reduce gene expression. siRNAs can be analysed by high-performance liquid chromatography (HPLC) as double-stranded duplex under non-denaturing conditions or as two single strands under denaturing conditions. Examination of the siRNA under non-denaturing conditions can be helpful to prove the success of duplex formation and detect the amount of excess single stranded RNA.
The analysis of siRNA is quite challenging due to their negatively charged phosphate backbone. Ion pair reversed phase liquid chromatography (IP-RP) is often the first choice for characterisation of such oligonucleotides. However, anion exchange chromatography (AEX) is also a popular method for oligonucleotide analysis. In this application note, the analysis of denatured and non-denatured siRNA by use of the bioinert YMC-Accura Triart Bio C18 column in IP-RP mode and the BioPro IEX QF column in AEX mode, is presented.
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