Quantitative miRNA expression analysis using fluidigm microfluidics dynamic arrays.
Author(s): Jang JS, Simon VA, Feddersen RM, Rakhshan F, Schultz DA, Zschunke MA, Lingle WL, Kolbert CP, Jen J
Publication: BMC Genomics, 2011, Vol. 12, Page 144
PubMed ID: 21388556 PubMed Review Paper? No
Purpose of Paper
Conclusion of Paper
Studies
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Study Purpose
The purpose of this study was to determine the effects of using FFPE lung specimens instead of frozen lung specimens on microRNA (miRNA) profiles measured using conventional and dynamic real-time PCR assays and microarrays. miRNA was extracted from xylene deparaffinized 10 uM sections of FFPE tissue using miRNeasy or RecoverAll. miRNA was extracted from frozen specimens using miRNeasy. miRNA was preamplified with pooled TaqMan assays.
Summary of Findings:
When real-time RT-PCR was conducted using 48.48 Fluidigm dynamic arrays, the average CT was only 12.48 compared to 16.21 using the ABI7900. The CT values were lower for frozen specimens than for the corresponding FFPE specimens, regardless of machine, but the data generated was highly correlated (R2=0.87-0.98). A linear relationship between CT and input quantity was observed for FFPE and frozen specimens using the Fluidigm dynamic arrays. 33-35% of miRNAs were detected on the arrays and there was a correlation of 0.94 between frozen and FFPE specimens. The range in fold change in expression between frozen and FFPE specimens was larger (0-13 fold) using the Fluidigm dynamic arrays than using microarray (0-3.5 fold).
Biospecimens
Preservative Types
- Formalin
- Frozen
Diagnoses:
- Neoplastic - Carcinoma
Platform:
Analyte Technology Platform RNA DNA microarray RNA Real-time qRT-PCR Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Biospecimen Preservation Type of fixation/preservation Formalin (buffered)
Frozen
Real-time qRT-PCR Specific Targeted nucleic acid 7900 HT
Fluidigm dynamic array
Real-time qRT-PCR Specific Template/input amount 10 ng
25 ng
50 ng
100 ng