Fig 1.
The SPC-SBE and MS approach for multiplexed miRNA quantification.
Reverse transcription of miRNA using stem-loop primers is followed by co-amplification of cNDA and competitors with single base alterations. A library of SBE primers with distinct masses are extended by biotin-ddNTPs in a multiplexed SBE reactions. Two extension products are generated for each SBE primer, one for miRNA and one for its competitor. Then the extension products are purified in an SPC process and analyzed by MALDI-TOF MS. Area ratio of extension product peaks is used to determine the levels of miRNAs.
Table 1.
Sequences of stem-loop RT primers (upper) and competitors (lower).
Fig 2.
Calibration curve established to calculate the initial miRNA concentration in the competitive PCR reaction.
Table 2.
SBE (Single Base Extension) primers.
Fig 3.
Mass spectrum for multiplexed analysis of four miRNA levels.
Levels of cellular and spiked miRNA are determined using the peak area ratio and the initial concentration of the corresponding competitors.
Table 3.
Peak area ratios measured from MS, calculated concentrations of miRNAs in cPCR reaction, and relative levels of miRNA measured in RT-qPCR.