GSE67075 ( miRNA )

colorectal cancer ( colorectal )

Real-time quantitative miRNA PCR analysis of human plasma samples; circulating miRNAs miR-34a and miR-150 associated with colorectal cancer progression

Cancer : 18 / Control : 30

Each miRNA was normalised by the ΔΔCt method using the average within sample Ct value. This technique involves the use of the mean expression value of all expressed microRNAs in a given sample as a normalisation factor for microRNA real-time quantitative PCR data. Thus the average within sample Ct value for each card is calculated by averaging all miRNA Ct values for each individual sample. This was performed using the Bioconductor package HTqPCR (www.bioconductor.org). The non-parametric Kruskal-Wallis test was used to determine between group variations by rank as the data was not normally distributed. A Wilcoxon rank sum test was subsequently used to perform pair-wise comparisons between the 5 groups for the significant miRNAs identified by the Kruskal-Wallis test. The file "fold_change_data.txt" is available on the series record and reports test/control (i.e., polyp/control) ratios.

The expression of 667 miRNAs was assessed in a discovery set of 48 plasma samples comprising normal, polyp, adenoma, and early and advanced cancer samples. Three miRNAs (miR-34a, miR-150, and miR-923) were further examined in a validation cohort of 97 subjects divided into the same five groups, and in an independent public dataset of 40 CRC samples and paired normal tissues.

qPCR miRNA expression profiling. Plasma samples from 48 donors comprising 8 normal, 8 polyp, 16 adenoma samples, 8 early stage cancer samples (stage I/II), and 8 advanced cancer samples (stage III/IV) were used. RNA was extracted from equal volumes of plasma from each donor.

Aherne ST, Madden SF, Hughes DJ, Pardini B et al. Circulating miRNAs miR-34a and miR-150 associated with colorectal cancer progression. BMC Cancer 2015 Apr 30;15:329. PMID: 25924769

Total RNA was isolated from 60µl of each plasma sample using the miRNeasy mini kit (Cat no 217004, Qiagen). The Qiagen supplementary protocol (Purification of total RNA, including small RNAs, from serum or plasma) was utilised with the following modifications: thawed plasma samples were centrifuged at 1000 x g for 5 minutes at 4ºC to remove excess debris from samples, RNA was extracted from the upper 50µl of each sample. To elute the RNA, 50µl of nuclease-free water was added to each spin column and incubated for 1 minute at room temperature before centrifuging into non-stick RNase-free microfuge tubes (Cat no AM12350, Ambion) to elute the RNA.

total RNA

FAM

Reverse transcription and quantitative PCR (qPCR) were performed on equal volumes of RNA from each sample according to the manufacturer's instructions using TaqMan® MicroRNA Reverse Transcription Kit (Cat no 4366596, Applied Biosystems) and Megaplex RT Primers to convert the miRNAs to cDNA, TaqMan® PreAmp Master Mix (Cat no 4391128, Applied Biosystems) and Megaplex PreAmp Primers for a preamplification step before real-time analysis. qPCR was performed using TaqMan® Universal Master Mix II, no UNG (Cat no 4440048, Applied Biosystems) on the 7900HT Fast Real-Time PCR system (Applied Biosystems).

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