Dataset Information

Purification of HIV RNA from serum using a polymer capture matrix in a microfluidic device.

ABSTRACT: In this report, we demonstrate the purification of DNA and RNA from a 10% serum sample using an oligonucleotide capture matrix. This approach provides a one-stage, completely aqueous system capable of purifying both RNA and DNA for downstream PCR amplification. The advantages of utilizing the polymer capture matrix method in place of the solid-phase extraction method is that the capture matrix eliminates both guanidine and the 2-propanol wash that can inhibit downstream PCR and competition with proteins for the binding sites that can limit the capacity of the device. This method electrophoreses a biological sample (e.g., serum) containing the nucleic acid target through a polymer matrix with covalently bound oligonucleotides. These capture oligonucleotides selectively hybridize and retain the target nucleic acid, while the other biomolecules and reagents (e.g., SDS) pass through the matrix to waste. Following this purification step, the solution can be heated above the melting temperature of the capture sequence to release the target molecule, which is then electrophoresed to a recovery chamber for subsequent PCR amplification. We demonstrate that the device can be applied to purify both DNA and RNA from serum. The gag region of HIV at a starting concentration of 37.5 copies per microliter was successfully purified from a 10% serum sample demonstrating the applicability of this method to detect viruses present in low copy numbers.

SUBMITTER: Root BE

PROVIDER: S-EPMC3270936 | biostudies-literature | 2011 Feb

REPOSITORIES: biostudies-literature

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Purification of HIV RNA from serum using a polymer capture matrix in a microfluidic device.

Root Brian E BE Agarwal Abhishek K AK Kelso David M DM Barron Annelise E AE

Analytical chemistry 20110107 3

In this report, we demonstrate the purification of DNA and RNA from a 10% serum sample using an oligonucleotide capture matrix. This approach provides a one-stage, completely aqueous system capable of purifying both RNA and DNA for downstream PCR amplification. The advantages of utilizing the polymer capture matrix method in place of the solid-phase extraction method is that the capture matrix eliminates both guanidine and the 2-propanol wash that can inhibit downstream PCR and competition with ...[more]

PMID: 21214255

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Project description:Cancer cells metastasize through the bloodstream either as single migratory circulating tumor cells (CTCs) or as multicellular groupings (CTC-clusters). Existing technologies for CTC enrichment are designed primarily to isolate single CTCs, and while CTC-clusters are detectable in some cases, their true prevalence and significance remain to be determined. Here, we developed a microchip technology (Cluster-Chip) specifically designed to capture CTC-clusters independent of tumor-specific markers from unprocessed blood. CTC-clusters are isolated through specialized bifurcating traps under low shear-stress conditions that preserve their integrity and even two-cell clusters are captured efficiently. Highly parallel architecture of the chip allows deterministic screening of clinically relevant volumes of blood samples at slow, and hence, non-damaging flow rates. Using the Cluster-Chip, we identify CTC-clusters in 30-40% of patients with metastatic cancers of the breast, prostate and melanoma. RNA sequencing of CTC-clusters confirms their tumor origin and identifies leukocytes within the clusters as being tissue-derived macrophages. Together, the development of a device for efficient capture of CTC-clusters will enable detailed characterization of their biological properties and role in cancer metastasis. We used the Cluster-Chip to capture CTC-clusters from the blood of a breast cancer patient with high CTC counts, released CTC-clusters in solution, stained them with TexasRed-conjugated antibodies against the leukocyte cell surface markers CD45, CD14 and CD16, and then isolated intact CTC-clusters individually using a micromanipulator. From a single time point, we retrieved 15 CTC-clusters, and each of those clusters was individually subjected to RNA-sequencing analysis using a next generation platform (SOLiD 5500). In addition, two leukocytes were isolated from the blood of a healthy donor were individually subjected to RNA-sequencing analysis using the same platform.

Dataset Information

Purification of HIV RNA from serum using a polymer capture matrix in a microfluidic device.

Publications

Purification of HIV RNA from serum using a polymer capture matrix in a microfluidic device.

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