Improve Yield and Quality from RNA and miRNA Extractions from FFPE Tissue
Monday, November 18, 2013
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Chapters
Introduction
FFPE Extraction Solutions using Agencourt FormaPure
Nucleic Acid Isolation From FFPE Tissue
To Sum up FFPE
Extractions from FFPE
FormaPure Overview
SPRI Bead Chemistry Overview
How Does SPRI Work?
FormaPure Protocol
FormaPure Automation
Obstacles Extracting from FFPE
Factors Affecting Nucleic Acid Quality & Yield
Crosslinking in FFPE
PCR with Crosslinking
Extraction from Different FFPE Tissues
Consistent Quality
Tips to Get the Highest Quality & Yield
Overcoming Obstacles During Extraction
Highly Efficient MicroRNA and Total RNA Purification from Formalin-Fixed Paraffin-Embedded Tissues (FFPE) using FormaPure Kits
Outline
What is micro-RNA (miRNA), and why is it important?
Pathway of miRNA biogenesis
Question: Can the Agencourt SPRI chemistry be optimized for miRNA purification?
Experimental Summary
The yield of the 22 base oligonucleotides using different concentrations of 100% Isopropanol in the binding steps
The yield of the 22 base oligonucleotides with different washing conditions
The yield from the manual extraction method is comparable to the automated extraction method
FormaPure miRNA extraction process overview
TaqMan let-7c miRNA assay for FFPE samples
Binding buffer conditions affect miRNA recovery: let-7c expression at the condition 1& 2 binding buffer
Amplification plots show expression of let-7c miRNA from manually extracted FFPE samples
Transfer the protocol on the Biomek FXp
Amplification plots show expression of let-7c miRNA from the Biomek extracted FFPE samples
Comparing the FormaPure Kit and miRNeasy FFPE Kit
Comparison of the RNA yield: FormaPure Kit gave a higher RNA yield than miRNeasy Kit
RNA profiles show typical fragmented FFPE RNA
Comparison of the RNA yield: FormaPure Kit gave a higher RNA yield than miRNeasy Kit.
RNA profiles show typical fragmented FFPE RNA
Comparison of the miRNA efficiency: FormaPure showed a higher miRNA extraction efficiency about 1/5 as much RNA was needed from FormaPure.
Comparison of the RNA purity: similar RNA purity and quality were produced by both FormaPure and miRNeasy FFPE Kits.
Summary
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