Areas between 8000 and 13000 μm2 of normal tissue, tumor tissue and tumor stroma were isolated by laser dissection from FFPE sections of human colon (A). Total RNA was extracted and miRNA levels were quantified using miRCURY LNA miRNA PCR (B). Normalized data are shown on a log2 scale as relative expression compared to normal tissue. hsa-miR-103 and hsa-let-7a were used as reference genes.
miRCURY LNA miRNA PCR Primers and commercially available DNA-based primer sets were used for real-time PCR amplification of serially diluted cDNA (106–10 copies). LNA-based primer sets showed superior sensitivity in all cases, especially for AT-rich sequences, such as hsa-miR-155 (61% AT) and hsa-miR-1 (73% AT).
Normalized expression levels of eight different miRNAs in two serum samples are shown. Total RNA purified from the equivalent of 8 µl serum was used in the RT reaction. hsa-let-7a and hsa-miR-103 were used as reference genes for normalization.
The miRCURY LNA miRNA PCR System uses one single cDNA synthesis reaction for all amplifications, reducing pipetting and saving time and sample. Two LNA-enhanced miRNA-specific qPCR primers enable highly specific and sensitive amplification and single nucleotide discrimination. The fast and easy workflow takes only 3 hours with minimal hands-on steps.
A poly(A) tail is added to the mature miRNA template (step 1A). cDNA is synthesized using a Poly(T) primer with a 3’ degenerate anchor and a 5’ universal tag (step 1B). The cDNA template is then amplified using two miRNA-specific and LNA-enhanced forward and reverse primers (step 2A). SYBR Green is used for detection (step 2B).