Table 1.
Sequences of matured small RNA molecules investigated. All sequences are in written in 5′-3′ direction.
Table 2.
Sequences of oligonucleotides used for qPCR measurements. UPL probe number 21 was used for the measurements.
Figure 1.
Schematic description of small RNA specific UPL-based quantitative PCR assay and our oligo design system.
Two steps small RNA specific UPL-based quantitative PCR assay relies on reverse transcription using small RNA specific stem-loop RT primer and real-time quantitative PCR reaction using small RNA specific forward primer, UPL21 probe and universal reverse primer (A). Workflow of our oligo design system (B). Primers and probe for the designed hsa-mir-181a specific UPL-based quantitative PCR assay (C).
Figure 2.
Validation of miRNA specific UPL-based quantitative PCR assays.
Arabidopsis thaliana specific miR168 expression in TCV infected leaves compared to mock control investigated by Northern-blot. As normalizer we used ethidium bromide staining of ribosomal RNA (A) Same samples as in panel “A” were measured with miRNA specific UPL-based quantitative PCR assays. As normalizer we used snoR41Y specific assays. (B). Quantification of LPS induced mir-155 level in mouse macrophages by both UPL-based qPCR system and mature miRNA specific qPCR assays of Life Technologies. As normalizer we used sno202 specific assays. (C).
Figure 3.
Sensitivity and specificity of miRNA specific UPL-based quantitative PCR system.
Amplification plot of mmu-mir-1 in range from 10 ng to 10–3 ng input mouse heart total RNA (A). Sequence similarities and differences between mir-181a, b, and c (B). Amplification plot of synthetic mir-181a miRNA ranging from 10 pM to 10–4 pM input mir-181a amplicon (C). Standard curve of synthetic mir-181a miRNA (D). Specificity and relative detection capacity of mir-181 specific UPL-based qPCR assays. Numbers represent the percentage of the signals measured on the synthetic amplicons. 100% is always the signal measured by an assay on its specific synthetic amplicon, like mir-180a assay on the mir-181a synthetic amplicon. In brackets the corresponding Cp values are shown.(E).
Figure 4.
Quantification of miRNA expression from FFPE samples.
Cancerous tissue and surrounding normal tissue obtained from microdissected human FFPE breast cancer sample [invasive duct carcinoma]. Images are showing hematoxylin-eosin stained sections with 10× and 40× original magnifications, respectively (A). Amplification plot of hsa-mir-155 in tumor tissue and normal tissue (B). Fold expression of RNU43 normalized hsa-mir-155 expression in tumor tissues relative to surrounding non-tumorous tissues from four independent breast cancer derived specimens. Error bars represent the standard deviations of the triplicate qPCR measurements (C).
Figure 5.
Measurement of siRNA expression with small RNA specific UPL-based quantitative PCR assays.
Sequence specificity of PRMT1 specific siRNA for exon 3 of PRMT1 (A). PRMT1 specific siRNA levels as detected by qPCR and the corresponding mPRMT1 mRNA as well as protein levels detected by qPCR and Western blot analysis (B).