Total RNA was purified from 5 µl human serum using the miRNeasy Serum/Plasma Kit. cDNA was then prepared from 0.7 µl serum equivalence (SE) using the miScript II RT Kit with miScript HiSpec Buffer. cDNA (0.7 µl SE) was used directly for miRNA profiling or one-tenth of the cDNA preparation (0.07 µl SE) was preamplified using the miScript PreAMP PCR Kit with Serum & Plasma miScript PreAMP Pathway Primer Mix prior to profiling. miRNA profiling was performed with the with the Plasma & Serum miScript miRNA PCR Array. Plots of [A] mean CT values achieved and [B]number of miRNAs detected demonstrate highly superior results from 10 fold less starting cDNA due to preamplification.
cDNA was prepared from a human universal reference RNA sample using the miScript II RT Kit with miScript HiSpec Buffer. Three different users performed preamplification using the miScript PreAMP PCR Kit and miFinder miScript PreAMP Pathway Primer Mix. Preamplified cDNA was used for miRNA profiling in duplicate with the miFinder miScript miRNA PCR Array. High levels of correlation were observed between users when CT values were [A] compared on a scatter plot or [B] plotted for all 3 users.
Preamplified cDNA and nonpreamplified cDNA from the same prep were used for miRNA profiling. Scatter plots of x-fold expression change calculations (2-ΔΔCT) between normal and tumor sections demonstrate high correlation between nonpreamplified and preamplified samples. [A] 96-plex miFinder miScript PreAMP Pathway Primer Mix and Array or [B] 384-plex miScript PreAMP miRNome Primer Mix and Array were used. The miRNeasy FFPE Kit was used to purify RNA from normal and tumor lung tissue 5 µM FFPE sections. cDNA was prepared from 10 ng total RNA using the miScript II RT Kit with miScript HiSpec Buffer and preamplified using the miScript PreAMP PCR Kit.
Preamplified cDNA and nonpreamplified cDNA from the same prep were used for miRNA profiling. Scatter plots of ΔCT values (normalized against housekeeping controls) between normal and tumor sections demonstrate high correlation between nonpreamplified and preamplified samples. [A] 96-plex miFinder miScript PreAMP Pathway Primer Mix and Array or [B] 384-plex miScript PreAMP miRNome Primer Mix and Array were used. The miRNeasy FFPE Kit was used to purify RNA from normal and tumor lung tissue 5 µM FFPE sections. cDNA was prepared from 10 ng total RNA using the miScript II RT Kit with miScript HiSpec Buffer and preamplified using the miScript PreAMP PCR Kit.