Analyzing miRNAs in ductal adenocarcinomas of the pancreas.
BACKGROUND
MicroRNAs (miRNAs) have gained attention as an epigenetic component involved in the development of pancreatic ductal adenocarcinoma (PDAC). Several methods for miRNA profiling are in common use, but the validity of these methods is not defined. The aim of this study was to define the optimal method for miRNA detection in PDAC.


METHODS
miRNA expression was determined using different and partially redundant methods (miRNA microarray, TaqMan low density array (TLDA), single tube quantitative RT-PCR). The data from different methods were statistically evaluated and tested for intermethodic consistency and reliability of the results. Finally, the miRNA expression status and the cell lines' ability to metastasize were correlated.


RESULTS
Comparing low and high metastatic cells, miRNA-microarrays identified fewer differentially expressed and only upregulated miRNAs (n=27; 27 up-regulated) compared with TLDAs (n=54; 19 up- and 35 down-regulated). Evaluating miRNAs that target tumor suppressor genes, expression of all single tube quantitative real-time reverse transcriptase PCR (qRT-PCR) validated miRNAs was detected to be significantly altered in TLDA analysis (100%). MiRNA microarrays detected only 25% of qRT-PCR validated miRNAs. Furthermore, results from TLDA analysis correlated well with data from qRT-PCR and presented ΔΔCt values from 3.5±1.86 (range 0.8-5.62) compared with 3.74±1.86 (range 0.78-5.95) in qRT-PCR.


CONCLUSIONS
Notable differences comparing data obtained from different screening methods were found. While TLDA and qRT-PCR correlated well in quantity and quality of the measured miRNAs, several tumor suppressor gene targeting and down-regulated miRNAs were not detected by miRNA-microarrays. This heterogeneity shows that care must be exercised when comparing results from different methods in PDAC.
Mees
Soeren Torge
ST
Department of General and Visceral Surgery, University Hospital of Muenster, Muenster, Germany. soerentorge.mees@ukmuenster.de
Schleicher
Christina
C
Mardin
Wolf Arif
WA
Senninger
Norbert
N
Colombo-Benkmann
Mario
M
Haier
Joerg
J
D000230:Adenocarcinoma / Q000378:metabolism* / Q000473:pathology; D021441:Carcinoma, Pancreatic Ductal / Q000378:metabolism* / Q000473:pathology; D045744:Cell Line, Tumor; D044127:Epigenesis, Genetic; D020869:Gene Expression Profiling; D006801:Humans; D035683:MicroRNAs / Q000378:metabolism*; D046228:Microarray Analysis; D010190:Pancreatic Neoplasms / Q000378:metabolism* / Q000473:pathology; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction
D035683:MicroRNAs