Quantitative Analysis of miRNA Expression

Total RNAs were isolated from patient samples and human cells with Trizol reagent, and quantified using a NanoDrop spectrophotometer (Thermo Scientific, Rockford, IL). Regular and stem-loop reverse transcription of mature miR-1291 were conducted as described previously [30 (link), 60 (link)]. RT-qPCR was performed on a MyIQ real-time PCR system (Bio-Rad, Hercules, CA). The cycle number (C_T) at which the amplicon concentration crossed a defined threshold was determined for each individual miRNA. The relative level of each analyte over internal standard (glyceraldehyde-3-phosphate dehydrogenase, or U6) was calculated as 2^−ΔΔCT, where ΔΔC_T = ΔC_{T treatment group} (analyte – internal standard) – ΔC_{T control group} (analyte – internal standard).

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Tu M.J., Pan Y.Z., Qiu J.X., Kim E.J, & Yu A.M. (2016). MicroRNA-1291 targets the FOXA2-AGR2 pathway to suppress pancreatic cancer cell proliferation and tumorigenesis. Oncotarget, 7(29), 45547-45561.

Publication 2016

NanoDrop MyIQ real-time PCR system

Cells Glyceraldehyde 3 phosphate dehydrogenase Human Mirna Patient Reverse transcription Rnas Stem Trizol

Corresponding Organization :

Other organizations : University of California, Davis, University at Buffalo, State University of New York, Roswell Park Comprehensive Cancer Center

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Variable analysis

independent variables

None explicitly mentioned

dependent variables

Relative level of each analyte (miR-1291) over internal standard (GAPDH or U6)

control variables

Total RNA isolation and quantification method (Trizol and NanoDrop)
Reverse transcription method (regular and stem-loop)
RT-qPCR system (MyIQ real-time PCR system)

controls

Positive control: None mentioned
Negative control: None mentioned

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