Small nucleolar RNA U91 is a new internal control for accurate microRNAs quantification in pancreatic cancer.
BACKGROUND
RT-qPCR quantification of miRNAs expression may play an essential role in pancreatic ductal adenocarcinoma (PDAC) diagnostics. RT-qPCR-based experiments require endogenous controls for the result normalization and reliability. However, expression instability of reference genes in tumors may introduce bias when determining miRNA levels.


METHODS
We investigated expression of 6 miRNAs, isolated from FFPE samples of pancreatic adenocarcinomas. Four internal controls were utilized for RT-qPCR result normalization: artificial miR-39 from C. elegans, U6 snRNA, miR-16 and snoRNA U91.


RESULTS
We found miR-21, miR-155 or miR-217 expression values in tumors may differ up to several times, depending on selected internal controls. Moreover, different internal controls can produce controversial results for miR-96, miR-148a or miR-196a quantification. Also, expression of our endogenous controls varied significantly in tumors. U6 demonstrated variation from -1.03 to 8.12-fold, miR-16 from -2.94 up to 7.38-fold and the U91 from -3.05 to 4.36-fold respectively. On the other hand, the most stable gene, determined by NormFinder algorithm, was U91. Each miRNA normalized relatively to the spike or U91, demonstrated similar expression values. Thus, statistically significant and insignificant differences between tumors and normal tissues for miRNAs were equal for the spike and the U91. Also, the differences between the spike and U91 were statistically insignificant for all of miRs except miR-217. Among three endogenous controls, U91 had the lowest average expression values and standard deviation in cancer tissues.


CONCLUSIONS
We recommend U91 as a new normalizer for miRNA quantification in PDACs.
Department of Pathology, Third Faculty of Medicine, Charles University in Prague and University Hospital Kralovske Vinohrady, Srobarova 50, 100 00, Prague 10, Czech Republic. alexey.popov@lf3.cuni.cz.
Alexey
A
Popov
Department of Pathology, Third Faculty of Medicine, Charles University in Prague and University Hospital Kralovske Vinohrady, Srobarova 50, 100 00, Prague 10, Czech Republic. arpad.szabomudr@yahoo.com.
Arpad
A
Szabo
Department of Pathology, Third Faculty of Medicine, Charles University in Prague and University Hospital Kralovske Vinohrady, Srobarova 50, 100 00, Prague 10, Czech Republic. vaclav.mandys@fnkv.cz.
Václav
V
Mandys
D000230:Adenocarcinoma / Q000235:genetics*; D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D000704:Analysis of Variance; D014408:Biomarkers, Tumor / Q000235:genetics; D016022:Case-Control Studies; D005260:Female; D020869:Gene Expression Profiling; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D008297:Male; D035683:MicroRNAs / Q000032:analysis* / Q000378:metabolism; D008875:Middle Aged; D010190:Pancreatic Neoplasms / Q000235:genetics*; D020537:RNA, Small Nucleolar / Q000378:metabolism*; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*
D014408:Biomarkers, Tumor; C524942:MIRN155 microRNA, human; C524940:MIRN21 microRNA, human; C553315:MIRN217 microRNA, human; D035683:MicroRNAs; D020537:RNA, Small Nucleolar