Viability of the IR laser-enabled laser capture microdissection (LCM) technique has been shown for a variety of different cell types. The procedure is remarkably simple and robust, and is available with Applied Biosystems® Arcturus® LCM instruments. Any Arcturus® LCM system can be used. The Arcturus® LCM method is extraordinarily gentle and ideal for microdissection of single cells or small numbers of cells. This system combines LCM and UV laser cutting for ultimate microdissection flexibility.
The PicoPure® DNA Extraction Kit allows you to extract and amplify DNA in the same tube, without organic extraction or spin columns, and to recover DNA from as few as 10 cells prepared by laser capture microdissection (LCM) or from milligrams of tissue. The PicoPure® DNA Extraction Kit provides conveniently packaged, stable proteinase K, PCR-compatible DNA Reconstitution Buffer, and a complete user guide. Each of 10 lyophilized proteinase K aliquots can be freshly reconstituted to make 150 µL of PicoPure® DNA Extraction Solution―enough for 150 x 10 µL CapSure® HS LCM Cap extractions or 30 x 50 µL CapSure® Macro LCM Cap extractions ( Figure 1).
|Figure 1. Reproducible DNA extraction and PCR amplification of multiple single-copy genes from ten laser capture microdissected cells. Cells were microdissected from human peripheral white blood cell cytospins, and DNA was extracted using 10 µL of PicoPure® DNA Extraction Solution. The extracts were analyzed directly in 35-cycle PCRs using primers for three single-copy genes.|
Because DNA is not lost due to organic extractions or spin columns, the PicoPure® kit enables reliable and reproducible DNA recovery from as few as 10 cells. PCR studies illustrate the advantages of the PicoPure® kit. Equal amounts of DNA were transferred to PCR, either directly in the PicoPure® kit buffer or after purification using a DNA extraction kit from a leading manufacturer. Quantitative real-time PCR (qRT-PCR) demonstrates that the PicoPure® kit samples amplify earlier, suggesting that there is more DNA present, and amplify more consistently, indicating less sample variation, than column-purified samples. The PicoPure® kit's reliably high DNA recovery enables increased detection sensitivity when amplifying single-copy genes from small samples in mutation analysis or genotyping assays. This high DNA recovery makes the kit ideal for small samples, such as those prepared by LCM.
DNA extracted with the PicoPure® kit is compatible with end-point PCR performed in the same 0.5 mL tube used for DNA extraction. For gene copy number quantitation, DNA from very small samples can be directly amplified by qRT-PCR without further purification, using platforms such as the StepOnePlus™ Real-Time PCR System. For highly sensitive mutation and genotype analysis, DNA can be extracted and directly amplified from small samples, and gene fragments can be separated by dHPLC (denaturing high-performance liquid chromatography), using platforms such as the WAVE® System (Transgenomics) (Figures 2 and 3).
|Figure 2. PicoPure® DNA extraction and quantitation of Her2/neu gene copy number by real-time PCR. 10, 100, and 1,000 SKBR3 cells were microdissected from cell smears using LCM.DNA was extracted using 10 µL of PicoPure® DNA Extraction Solution and, without further purification, all 10 µL was used in a 20 µL real-time PCR reaction. The number of gene copies per sample was determined and divided by the number of cells per sample to determine the number of gene copies per cell.|
Figure 3. PicoPure® DNA Extraction Solution is compatible with endpoint PCR and amplicon detection using dHPLC. DNA was extracted from 10 peripheral white blood cells per sample using the PicoPure® DNA Extraction Kit. This DNA was used, without further purification, in PCR to amplify a 125 bp human p53 gene fragment and a 245 bp human cystic fibrosis gene (CFTR) fragment. The PCR products were then separated and detected using the dHPLC WAVE® System. Data courtesy of Scott Hamlin and Nicolas Neckelmann, Transgenomics, Inc., San Diego, CA.
The PicoPure® DNA Extraction Kit enables successful recovery of genomic DNA from animal tissue sections and cell samples prepared using a wide range of methods.* Superior results are obtained from formalin-fixed, paraffin-embedded (FFPE) tissue sections, frozen tissue sections, ethanol-fixed cells, and cytological smears (Figure 4).
|Figure 4. PicoPure® DNA extraction and b-globin gene amplification from LCM samples prepared by various methods. Cell and tissue samples were prepared for LCM using the methods listed below. LCM was used to collect samples. DNA was extracted using the PicoPure® DNA Extraction Kit, a single-copy 536 bp human b-globin fragment was amplified from each sample using standard protocols, and the products were electrophoresed on a 10% polyacrylamide/TBE gel and stained with SYBR® Gold Nucleic Acid Stain. M: DNA marker. Lanes 1–3: ethanol-fixed SKBR3 cells. Lanes 4–6: FFPE breast tissue. Lanes 7–9: frozen, ethanol-fixed foreskin tissue. Lane 10: positive PCR control of 1 ng human genomic DNA template. Lane 11: negative control, no PCR template.|
For Research Use Only. Not for use in diagnostic procedures.