Multiplex RT-PCR for miRNA Profiling

We used a fixed volume of 3 μl of total purified for the reverse transcription (RT). A set of 754 miRNAs were processed in CSF samples, using TaqMan miRNA Reverse Transcription Kit and Multiplex RT Assays (Human pool sets A and B) (Applied Biosystems) as previously described [18 (link)]. For RNA isolated from plasma samples, we used custom RT primer pool directed to the miRNAs identified as altered in CSF following the appropriate protocol. Each protocol has demonstrated sensitivity, specificity as well as repeatable and accurate results [19 (link)]. RT products for both CSF and plasma were then preamplified by means of TaqMan PreAmp Master Mix and/or Megaplex PreAmp Primers (Human pool sets A and B) (Applied Biosystems), or by custom preamplification primer pool, respectively. Preamplification step is mandatory before real-time PCR (RT-PCR) reaction when analytical sensitivity is the utmost importance and the sample is limiting [18 (link)].