br and condensed with high purity for
and condensed with high purity for the following miRNA quanti-fication as a DPD probe (Fig. 4B).
3.2. Characterization of DNA-Peptide dendrimer probe
The obtained RP8-MAP4-DNA was analyzed using a LCQ Deca XP Plus ion trap MS to provide definitive evidence for its existence. The signal at 18982.2 Da was consistent with the theoretical mass value of RP8-MAP4-DNA (Fig. 4C). In addition, the conjugated DNA did not have impact on the C12FDG efficiency. By adding trypsin to equal amounts of RP8-MAP4-DNA and RP8-MAP4, there was no significant difference in the released amount of reporter peptide (Data not shown).
For miRNA detection probe, its hybridization efficiency and specificity with target miRNA are critical factors contributing to its performance. In this study, the hybridization efficiency of RP8-MAP4-DNA was calculated by comparing the amount of hybrid-ized RP8-MAP4-DNA to the amount of immobilized target miR-21. In addition, factors including ionic strength, temperature and time were optimized to maximize the hybridization efficiency. As a result, a buffer containing 10 mM Tris, 100 mM KCl, 1 mM MgCl2, pH 7.4 was selected. Hybridization temperature of 45 C and hybridi-zation time of 4 h provided the highest efficiency (Fig. S8). Under these conditions, hybridization efficiency can achieve ~92.1%.
Probe specificity was also evaluated, due to the high degree of similarity between miRNA sequences . Some miRNA family members vary by a single nucleotide. Thus, single or double mis-matched miRNAs were employed in parallel with miR-21. Relative hybridization efficiency was worked out by comparing the reporter peptide amounts of perfectly matched and mismatched targets. The
Fig. 4. Size exclusion chromatograms of (A) RP8-MAP4, DNA and their products after conjugation and (B) purified RP8-MAP4-DNA at l ¼ 220 nm. The mobile phase consisted of 150 mM phosphate buffer containing 2 M sodium chloride (pH ¼ 7.0). The conditions of size exclusion chromatography were described in supplementary material. (C) Full scan spectrum of the DPD probe RP8-MAP4-DNA. Mass spectrometry detection was performed on a Thermo LCQ Deca XP Plus ion trap MS, which was operated with electrospray ionization in the positive mode.
3.3. Development and validation of sensitive miRNA quantification assay combining DNA-Peptide dendrimer probe and LC-MS/MS
To quantify target miRNA, all the miRNA samples were bio-tinylated in advance and then immobilized on the streptavidin agarose beads. In our previous study, the maximum loading ca-pacity of streptavidin agarose beads for biotinylated miRNAs was ~2.1 biotin molecules/streptavidin , so almost all the miRNAs in samples can be immobilized using excessive agarose beads.
Under the optimized conditions for DPD probe and LC-MS/MS, the signal intensity of miR-21 was ~8 times greater compared to that without signal amplification . Then, standard curves were constructed from the relative peak area ratio between the reporter peptide and the stable isotope-labeled internal standard across the concentration (1/x2 weighting). From Fig. 5A, it can be seen that the area ratio is sensitive to the concentration of miR-21, the fitting
range is from 0.2 pM to 10 nM, and the LOQ was 0.2 pM (Fig. S9). In addition, QC samples prepared in tRNA library yeast were used to evaluate the precision and the accuracy of the assay. Six replicates of QC samples prepared at four different miR-21 con-centration levels were evaluated in three validation runs. The re-sults are listed in Table 1S. Both accuracy and precision were
±15% (LLOQ, ±20%). In particular, the %RSD and %bias measured on the LLOQ was much less than 5% and generally better than 3%, indicating the good accuracy and precision at low concentrations . This feature may bring the benefits of early diagnosis and increase the diagnostic and theranostic accuracy.