Dataset Information

Exosomes molecular diagnostics: Direct conversion of exosomes into the cDNA for gene amplification by two-step polymerase chain reaction.

ABSTRACT: Exosomes are cell derived lipid nanoparticle with a size of 30-100 nm in diameter, found in almost all biological fluids. The composition of the exosomes is mainly lipid, proteins, RNA, DNA, and non-coding RNAs. Currently, most available methods and commercial kits for exosomal-RNA (Exo-RNA) isolation have limitations and shortcomings. Small starting volume of exosomes and the use of extraction/filtration columns results usually insufficient yield of exosomal RNA after isolation. The majority of RNA contained in purified exosomes range in size from 15-500 nucleotides. Some RNA isolation kits are well suited for small RNA transcripts isolation but larger mRNA transcripts are hard to detect. For all of the kits, the cost prize per sample analyzed is very high. Our current method provides a novel way for direct conversion of exosomes into cDNA synthesis (Exo-cDNA) and subsequent gene detection by polymerase chain reaction (PCR). This method has several advantages compared to established available kits. No extraction column is utilized in this procedure which means total recovery of exosomal RNA with maximal yield. In addition, this method is fast and uses a minimal amount of lab supplies, thereby reducing the overall working costs. Our findings suggest that direct conversion of exosomes into cDNA and subsequent gene amplification by two step PCR is a most efficient and reproducible technique. This novel method can be applied to and is useful to advance molecular research of exosomes by solving the problem of low molecular yields.

SUBMITTER: Haque S

PROVIDER: S-EPMC6706146 | biostudies-literature | 2018

REPOSITORIES: biostudies-literature

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Publications

Exosomes molecular diagnostics: Direct conversion of exosomes into the cDNA for gene amplification by two-step polymerase chain reaction.

Haque Shabirul S Vaiselbuh Sarah R SR

Journal of biological methods 20180725 3

Exosomes are cell derived lipid nanoparticle with a size of 30-100 nm in diameter, found in almost all biological fluids. The composition of the exosomes is mainly lipid, proteins, RNA, DNA, and non-coding RNAs. Currently, most available methods and commercial kits for exosomal-RNA (Exo-RNA) isolation have limitations and shortcomings. Small starting volume of exosomes and the use of extraction/filtration columns results usually insufficient yield of exosomal RNA after isolation. The majority of ...[more]

PMID: 31453246

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Project description:Polymerase chain reaction (PCR) is an in vitro technology in molecular genetics that progressively amplifies minimal copies of short DNA sequences in a fast and inexpensive manner. However, PCR performance is sensitive to suboptimal processing conditions. Compromised PCR conditions lead to artifacts and bias that downgrade the discriminatory power and reproducibility of the results. Promising attempts to resolve the PCR performance optimization issue have been guided by quality improvement tactics adopted in the past for industrial trials. Thus, orthogonal arrays (OAs) have been employed to program quick-and-easy structured experiments. Profiling of influences facilitates the quantification of effects that may counteract the detectability of amplified DNA fragments. Nevertheless, the attractive feature of reducing greatly the amount of work and expenditures by planning trials with saturated-unreplicated OA schemes is known to be relinquished in the subsequent analysis phase. This is because of an inherent incompatibility of ordinary multi-factorial comparison techniques to convert small yet dense datasets. Treating unreplicated-saturated data with either the analysis of variance (ANOVA) or regression models destroys the information extraction process. Both of those mentioned approaches are rendered blind to error since the examined effects absorb all available degrees of freedom. Therefore, in lack of approximating an experimental uncertainty, any outcome interpretation is rendered subjective. We propose a profiling method that permits the non-linear maximization of amplicon resolution by eliminating the necessity for direct error estimation. Our approach is distribution-free, calibration-free, simulation-free and sparsity-free with well-known power properties. It is also user-friendly by promoting rudimentary analytics. Testing our method on published amplicon count data, we found that the preponderant effect is the concentration of MgCl2 (p<0.05) followed by the primer content (p<0.1) whilst the effects due to either the content of the deoxynucleotide (dNTP) or DNA remained dormant (p>0.1). Comparison of the proposed method with other stochastic approaches is also discussed. Our technique is expected to have extensive applications in genetics and biotechnology where there is a demand for cheap, expedient, and robust information.

Dataset Information

Exosomes molecular diagnostics: Direct conversion of exosomes into the cDNA for gene amplification by two-step polymerase chain reaction.

Publications

Exosomes molecular diagnostics: Direct conversion of exosomes into the cDNA for gene amplification by two-step polymerase chain reaction.

Similar Datasets

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