Abstract
Abstract 4197
Background
Though primary cells obtained from patients are necessary to scientists, it has been problems that their amount is not sufficient to perform various assays and that the cell specimens are often impaired, or their constituents or products are destroyed during the transport to the scientists. To address these problems, we developed a bio-cell chip, a chip having hundreds kind of cells arrayed and immobilized on a small slide. We arrayed the cells in a tiny space by spotting them onto a supporting matrix, so that hundreds of assays can be conducted with only a small amount of sample and several hundreds kinds of samples can be assayed under the same condition. We developed the direct extraction of DNA from bio-cell chip and successfully amplified the extracted DNA. Here, we introduce simple and convenient method of DNA extraction utilizing bio-cell chip, which can handle hundreds of specimen at one time.
Methods
Peripheral blood anticoagulated with EDTA or sodium citrate were centrifuged at 2000 rpm for 20 minutes, and leukocyte layers were transferred to 1.5 ml tube by careful pipetting. Buffy coats are adjusted to the cell count of 1,000 cells/ μl to 10,000 cells/ μl with Phosphate Buffered Saline. One microliter of cell suspension are loaded onto each of 92 well bio cell-chip and air-dried. To investigate the duration of DNA stability of cells loaded on bio cell-chip, loaded bio cell-chip were stored at room temperature for 5day, 15day, and 1 month before DNA extraction. Cell-chip preparation was done as previously published. Briefly, the substrate for the bio cell-chip was a slide glass on which a 1 mm thick perforated poly- dimethylsiloxane (PDMS) layer was bonded to form 16×6 wells array for subsequent cell seeding. Each cylindrical shape with 1.5 mm in diameter and positioned within a lattice pattern with its own indexing number printed on the slide glass. Maximum volume that can be loaded in each cylinder was 10ul. For DNA extraction, 2 ul of BR-A buffer (GenScript BloodReadyTMMultiplex PCR System) was dropped onto each of the wells of bio-cell chip and dried cells in the bottom of wells were mixed with BR-A buffer solution thoroughly by pipetting up and down for 10-15 times. Then, BR-A buffer additionally dropped into each well up to 20ul volume finally and mixed well. One or multiple gene targets can be amplified using a pair of primers or multiple pairs of primers from the mixture of buffer with cells without DNA purification. Then, PCR Amplification was carried out using the MJ Research DNA thermal cycler programmed to denature DNA at 95°C for 30s, and 40cycles of annealing at 62°C and extension at 72°C for 30sec in a 20μl mixture containing 1U BioTherm‘DNA Polymerase (Genecraft, GERMANY), 1μl(10pM/ul) of each oligonucleotide primer, 2.5mM dNTP, and genomic DNA.
Results and conclusion
The PCR amplification results were successful using BioTherm‘DNA Polymerase. When we carried out PCR amplification using the PCR Premix (BloodReadyTM PCR System), we had no PCR products. The target PCR products were observed from genomic DNA sample with above 100 cells. Also, PCR amplification for genomic DNA from stored cell for 5day, 15 day, and 1 month was successful. With this extraction method, PCR was successful and not only minimize the simplification of experimental procedure but also save the time.
Disclosures:
No relevant conflicts of interest to declare.
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