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miRNA PCR Array and Assay Application Examples

Circulating Biomarker Discovery using the miScript PCR System


Total RNA was isolated from 200 Ál normal and lung cancer serum samples (3 each) using the miRNeasy Serum/Plasma Kit. RNA was eluted using 14 Ál nuclease free-water. For reverse transcription, one-tenth of the RNA eluate was reverse transcribed into cDNA using the miScript II RT Kit with miScript HiSpec Buffer. The Human Serum & Plasma 384HC miScript miRNA PCR Array, in combination with the miScript PCR System, was used to profile mature miRNA expression by real-time PCR. The miRNeasy Serum/Plasma Spike-In Control was used to calibrate the data, and a plate mean of commonly expressed miRNAs was used for normalization. In total, the expression of 183 mature miRNAs was detected. Included in the miRNAs that exhibited significant upregulation are hsa-miR-25 and hsa-miR-223, which have previously been shown to be blood-based markers for patients with lung cancer (1).
1. Chen X, et al. (2008) Characterization of microRNAs in serum: a novel class of biomarkers for diagnosis of cancer and other diseases. Cell Res. 18, 997.

High quality, rapid miRNA Expression Profiling using FFPE Samples


Total RNA was isolated from thin (5 Ám) normal lung and lung tumor FFPE tissue sections using the miRNeasy FFPE Kit. cDNA was then prepared from each total RNA in a miScript II reverse transcription reaction using HiSpec Buffer. In real-time PCR, the Human miFinder miScript miRNA PCR Array, in combination with the miScript SYBR Green PCR Kit, was used to profile mature miRNA expression. Data analysis was performed using the online miScript miRNA PCR Array data analysis tool. The raw data in Panel A (CT Values: FFPE Isolations 1, 2, and 3) demonstrates the high reproducibility that can be achieved using the miRNeasy FFPE Kit in combination with the miScript PCR System and miScript miRNA PCR Arrays. Each isolation was from a different FFPE tissue section, with all three normal lung samples coming from one donor. When normal and tumor tissue samples are compared in Panel B, a scatter plot of 2-ΔΔCT values demonstrates that significant differences exist between the mature miRNA expression levels of the two tissue types (Panel B, ▒ 3-fold [red lines] used as a cutoff for significance). The online miScript miRNA PCR Array data analysis tool automatically performs all ΔΔCT based fold-change calculations from your uploaded raw threshold cycle data and provides various statistical outputs such as a scatter plots, fold-regulation values, and volcano plots. Using the miRNeasy FFPE Kit, miScript PCR System, and miScript miRNA PCR Arrays, miRNA expression patterns in FFPE samples can be rapidly profiled using real-time PCR.

miScript Preamplification Unlocks miRNA Expression Profiles in Limiting Samples


Total RNA was isolated from 5 Ál aliquots of normal (n=3) and colorectal cancer (n=3) serum (each isolation was from a different serum donor) using the miRNeasy Serum/Plasma Kit. cDNA was then prepared from 0.7 Ál serum equivalence (SE) of each total RNA isolation in a miScript II reverse transcription reaction using HiSpec Buffer. Following reverse transcription, one-tenth of the cDNA preparation (0.07 Ál SE) was used in a miScript PreAMP reaction with the Human Serum & Plasma 384HC miScript PreAMP Primer Kit. From there, the preamplified cDNA was diluted 20-fold, and 1 Ál of diluted cDNA was used per well of the Human Serum & Plasma 384HC miScript miRNA PCR Array. This PCR array profiles the expression of 372 miRNAs detectable in human serum and plasma samples using the miScript PCR System. A scatter plot of 2-ΔCT values demonstrates that significant differences exist between the mature miRNA expression levels of the normal serum and colorectal cancer serum pools (Panel B, ▒ 2-fold [red lines] used as a cutoff for significance). Those targets that exhibited differential expression include novel miRNAs as well as miRNAs whose expression has been previously shown to be regulated in colorectal cancer (hsa-miR-19b, hsa-miR-29b, hsa-miR-106b, and hsa-miR-223)(1,2).

1. Arndt, G., L. Dossey, et al. (2009). "Characterization of global microRNA expression reveals oncogenic potential of miR-145 in metastatic colorectal cancer." BMC Cancer 9(374).

2. Volinia, S., G. Calin, et al. (2006). "A microRNA expression signature of human solid tumors defines cancer gene targets." PNAS 103(7): 2257-61.

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