Sample Preparation |
Rapid Extraction of PCR-Ready DNA and RNA
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Biocompare Review 2/19/09
Applications
- Isolation of DNA from FFPE samples for PCR-based analysis, e.g., microsatellite
detection, SNP detection, tumor heterogeneity studies, copy number detection,
methylation analysis, and Short Tandem Repeat (STR) analysis.
The analysis of nucleic acid from formalin-fixed, paraffin-embedded (FFPE)
specimens is challenging due to the extensive cross-linking of all tissue components
during the fixation process. These challenges include chemical modification
of the DNA, cross-linking of DNA with other molecules, degradation of the DNA,
and the limited amount of nucleic acid in the samples.
The QuickExtract™ FFPE DNA Extraction Kit is a fast, simple, and inexpensive
method for preparing genomic DNA for PCR amplification from archival samples
(Fig. 1). The protocol requires only heat treatment to melt the paraffin, lyse
the cells, decrease the fomalin-induced cross-linking in the sample, and degrade
compounds inhibitory to amplification. Following heat treatment, the sample
DNA is ready for PCR (Figs. 2 and 3).
Note: This product is only intended for PCR- and qPCR-based applications.
If DNA is required for other molecular biology applications, consider the MasterPure™ DNA
Purification Kit.
Benefits
- PCR-ready DNA in minutes, not days.
- No xylene or phenol extraction.
- No columns, transfers, or sample loss.
- Extracted DNA is compatible with both real-time and endpoint PCR.
Figure 1. Overview of the QuickExtract™ FFPE DNA extraction
procedure.
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Figure 2. PCR amplification of DNA from a slide-mounted,
FFPE preserved human skeletal muscle tissue section, extracted with the
QuickExtract™ FFPE DNA Extraction Kit. Two microliters of undiluted,
extracted DNA was amplified with primers for three different loci: tumor
protein 53 (TP53), dystrophin (DMD), and tumor necrosis factor (TNF). The
products were separated on a 3% agarose gel and were visualized with SYBR® Gold.
Lane M, 100-bp DNA ladder; lane 1, exon 2 of TP53; lane 2, exon 3 of TP53;
lane 3, exon 11 of TP53; lane 4, exon 6 of DMD; lane 5, exon 50 of DMD;
lane 6, exon 3 of DMD; lane 7, exon 4 of TNF. |
Figure 3. PCR amplification of DNA from slide-mounted,
FFPE-preserved, human tissue sections, combined and extracted with the QuickExtract™ FFPE
DNA Extraction Kit. Two microliters of undiluted, extracted DNA was
amplified with primers for seven different STR loci. The products were separated
on a 3% agarose gel and were visualized with SYBR® Gold. The expected
allele-to-allele variation in STR sequences results in multiple products
per primer set. Lane M, 100-bp DNA ladder; lane 1, RENA4; lane 2, D3S1358;
lane 3, D7S820; lane 4, THO1; lane 5, D19S253; lane 6, D21S11; lane 7, AMEL. |
FailSafe™ PCR System
MasterPure™ Complete DNA and RNA Purification Kit
MMLV Reverse Transcriptase 1st-Strand cDNA Synthesis Kit
MMLV High Performance Reverse Transcriptase
QuickExtract™ FFPE RNA Extraction Kit
TAQXpedite™ PCR System (FAST End-Point)
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