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Pathol Oncol Res. 2016 Jul;22(3):505-13. doi: 10.1007/s12253-015-0027-4. Epub 2015 Dec 17.

miRNA Isolation from FFPET Specimen: A Technical Comparison of miRNA and Total RNA Isolation Methods.

Author information

1
Cell Analysis Laboratory, 2nd Department of Internal Medicine, Semmelweis University, Szentkirályi Str. 46, Budapest, 1088, Hungary. nagyzsofiab@gmail.com.
2
Molecular Medicine Research Unit, Hungarian Academy of Sciences, Budapest, Hungary.
3
Cell Analysis Laboratory, 2nd Department of Internal Medicine, Semmelweis University, Szentkirályi Str. 46, Budapest, 1088, Hungary.

Abstract

MiRNA remain stable for detection and PCR-based amplification in FFPE tissue samples. Several miRNA extraction kits are available, however miRNA fraction, as part of total RNA can be isolated using total RNA purification methods, as well. Our primary aim was to compare four different miRNA and total RNA isolation methods from FFPE tissues. Further purposes were to evaluate quantitatively and qualitatively the yield of the isolated miRNA. MiRNAs were isolated from normal colorectal cancer FFPE specimens from the same patients. Two miRNA isolation kits (High Pure miRNA Isolation Kit, miRCURY™ RNA Isolation Kit) and two total RNA isolation kits were compared (High Pure RNA Paraffin Kit, MagNA Pure 96 Cellular RNA LV Kit). Quantity and quality were determined, expression analysis was performed by real-time PCR using qPCR Human Panel I + II (Exiqon) method detecting 742 human miRNAs in parallel. The yield of total RNA was found to be higher than miRNA purification protocols (in CRC: Ex: 0203 ± 0021 μg; HPm: 1,45 ± 0,8 μg; HPp: 21,36 ± 4,98 μg; MP: 8,6 ± 5,1 μg). MiRNAs were detected in lower relative quantity of total RNA compared to the miRNA kits. Higher number of miRNAs could be detected by the miRNA isolation kits in comparison to the total RNA isolation methods. (Ex: 497 ± 16; HPm: 542 ± 11; HPp: 332 ± 36; MP: 295 ± 74). Colon specific miRNAs (miR-21-5p;-34-5p) give satisfying results by miRNA isolation kits. Although miRNA can be detected also after total RNA isolation methods, for reliable and reproducible miRNA expression profiling the use of miRNA isolation kits are more suitable.

KEYWORDS:

Colon; Colorectal cancer; Comparison; Concentration; Ffpe; Isolation; Isolation kit; MiRNA; Normalization; Reference MiRNA; Total RNA; U6

PMID:
26678076
DOI:
10.1007/s12253-015-0027-4
[Indexed for MEDLINE]

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