miRNA Isolation from FFPET Specimen: A Technical Comparison of miRNA and Total RNA Isolation Methods.
Author(s): Nagy ZB, Wichmann B, Kalmár A, Barták BK, Tulassay Z, Molnár B
Publication: Pathol Oncol Res, 2016, Vol. 22, Page 505-13
PubMed ID: 26678076 PubMed Review Paper? No
Purpose of Paper
This paper compared the effects of using microRNA (miRNA, miR)-specific or general RNA isolation kits on the yield and quality of miRNA extracted from formalin-fixed paraffin-embedded (FFPE) colorectal specimens. The effect of RNA extraction kit and reference gene choice on the differential expression of miR-21 and miR-34 in tumor specimens was also investigated.
Conclusion of Paper
As expected, the total RNA isolation kits resulted in higher overall RNA yields than the miRNA-specific extraction kits, but the miRNA-specific kits resulted in isolation of more miRNA species and higher levels of miRNA. Based on RNA yield, the High Pure paraffin kit was the better RNA isolation kit and the High Pure miRNA kit was the better miRNA isolation kit, but expression levels of individual miRNA were strongly correlated between kits of the same type (general RNA and miRNA-specific). Importantly, elevated expression of miR-21and miR-34 in tumor relative to normal specimens was dependent on the reference gene chosen but not the extraction method used.
This study investigated the effects of using miRNA-specific or general RNA isolation kits on the yield and quality of miRNA extracted from FFPE colorectal specimens and the effects of extraction kit and reference gene choice on the differential expression of miR-21 and miR-34 in tumor specimens. This study included FFPE blocks stored less than 5 years from three patients that contained >60% tumor content and their matched normal adjacent tissue. For each extraction, three 10µm sections were placed in a tube and deparaffinized in two 10 min changes of xylene, washed in ethanol, and air-dried at 55˚C before extraction with each of the 4 kits. miRNA was quantified using the mercury Universal RT microRNA PCR and the Exiqon Ready-to-use microRNA PCR Human Panels I + II V2.
Summary of Findings:
RNA yields were higher using RNA isolation kits than miRNA isolation kits, but miRNA kits resulted in selected isolation of 20-200nt RNA. The High Pure RNA paraffin kit produced the highest yields of the RNA isolation kits and the High Pure miRNA isolation kit produced higher yields than the miRCURY RNA Isolation Kit. As expected, a larger number of miRNA were found and were at higher levels in specimens isolated using the miRNA-specific methods than the total RNA kits, but the two miRNA-specific methods were comparable. Levels of the miRNA were strongly correlated between the two miRNA-specific extraction methods (r2=0.8911) and between the two total RNA extraction kits (r2=0.8571), but were only modestly correlated between the miRCURY RNA kit and the two RNA specific kits (r2=0.5216 for the MagNAPure 96 kit and r2=0.6796 for the High Pure Paraffin kit). Of the three tested reference RNAs, U6 was not found in all specimens but the SNORD38B and SNORD49A were reliably detected in all specimens. Importantly, elevated levels of miR-21 and miR-34 in tumor specimens were detected regardless of RNA (or miRNA) extraction method when data was normalized to miR-490-3p, but the elevated levels in tumor specimens were less obvious when the data was normalized to SNORD38B.
- Neoplastic - Normal Adjacent
- Neoplastic - Carcinoma
Analyte Technology Platform RNA Fluorometry RNA Real-time qRT-PCR RNA Automated electrophoresis/Bioanalyzer
Classification Pre-analytical Factor Value(s) Analyte Extraction and Purification Analyte isolation method High Pure miRNA Isolation Kit
miRCURY RNA Isolation Kit
High Pure RNA Paraffin Kit
MagNA Pure 96 Cellular RNA LV Kit
Real-time qRT-PCR Specific Data handling Normalized to miR-490-3p
Normalized to SNORD38B
Real-time qRT-PCR Specific Targeted nucleic acid 742 miRNA panel