A comparison of RNA amplification techniques at sub-nanogram input concentration

<p>Abstract</p> <p>Background</p> <p>Gene expression profiling of small numbers of cells requires high-fidelity amplification of sub-nanogram amounts of RNA. Several methods for RNA amplification are available; however, there has been little consideration of the accuracy of these methods when working with very low-input quantities of RNA as is often required with rare clinical samples. Starting with 250 picograms-3.3 nanograms of total RNA, we compared two linear amplification methods 1) modified T7 and 2) Arcturus RiboAmp HS and a logarithmic amplification, 3) Balanced PCR. Microarray data from each amplification method were validated against quantitative real-time PCR (QPCR) for 37 genes.</p> <p>Results</p> <p>For high intensity spots, mean Pearson correlations were quite acceptable for both total RNA and low-input quantities amplified with each of the 3 methods. Microarray filtering and data processing has an important effect on the correlation coefficient results generated by each method. Arrays derived from total RNA had higher Pearson's correlations than did arrays derived from amplified RNA when considering the entire unprocessed dataset, however, when considering a gene set of high signal intensity, the amplified arrays had superior correlation coefficients than did the total RNA arrays.</p> <p>Conclusion</p> <p>Gene expression arrays can be obtained with sub-nanogram input of total RNA. High intensity spots showed better correlation on array-array analysis than did unfiltered data, however, QPCR validated the accuracy of gene expression array profiling from low-input quantities of RNA with all 3 amplification techniques. RNA amplification and expression analysis at the sub-nanogram input level is both feasible and accurate if data processing is used to focus attention to high intensity genes for microarrays or if QPCR is used as a gold standard for validation.</p

Reactivity-Dependent PCR: Direct, Solution-Phase in Vitro Selection for Bond Formation

In vitro selection is a key component of efforts to discover functional nucleic acids and small molecules from libraries of DNA, RNA, and DNA-encoded small molecules. Such selections have been widely used to evolve RNA and DNA catalysts and, more recently, to discover new reactions from DNA-encoded libraries of potential substrates. While effective, current strategies for selections of bond-forming and bond-cleaving reactivity are generally indirect, require the synthesis of biotin-linked substrates, and involve multiple solution-phase and solid-phase manipulations. In this work we report the successful development and validation of reactivity-dependent PCR (RDPCR), a new method that more directly links bond formation or bond cleavage with the amplification of desired sequences and that obviates the need for solid-phase capture, washing, and elution steps. We show that RDPCR can be used to select for bond formation in the context of reaction discovery and for bond cleavage in the context of protease activity profiling.Chemistry and Chemical Biolog

The Digital MIQE Guidelines Update: Minimum Information for Publication of Quantitative Digital PCR Experiments for 2020

Digital PCR (dPCR) has developed considerably since the publication of the Minimum Information for Publication of Digital PCR Experiments (dMIQE) guidelines in 2013, with advances in instrumentation, software, applications, and our understanding of its technological potential. Yet these developments also have associated challenges; data analysis steps, including threshold setting, can be difficult and preanalytical steps required to purify, concentrate, and modify nucleic acids can lead to measurement error. To assist independent corroboration of conclusions, comprehensive disclosure of all relevant experimental details is required. To support the community and reflect the growing use of dPCR, we present an update to dMIQE, dMIQE2020, including a simplified dMIQE table format to assist researchers in providing key experimental information and understanding of the associated experimental process. Adoption of dMIQE2020 by the scientific community will assist in standardizing experimental protocols, maximize efficient utilization of resources, and further enhance the impact of this powerful technology

PCR-Based Methods for the Enrichment of Minority Alleles and Mutations

BACKGROUND: The ability to identify low-level somatic DNA mutations and minority alleles within an excess wild-type sample is becoming essential for characteriz-ing early and posttreatment tumor status in cancer pa-tients. Over the past 2 decades, much research has fo-cused on improving the selectivity of PCR-based technologies for enhancing the detection of minority (mutant) alleles in clinical samples. Routine applica-tion in clinical and diagnostic settings requires that these techniques be accurate and cost-effective and re-quire little effort to optimize, perform, and analyze. CONTENT: Enrichment methods typically segregate by their ability to enrich for, and detect, either known or unknownmutations. Although there are several robust approaches for detecting known mutations within a high background of wild-typeDNA, there are few tech-niques capable of enriching anddetecting low-level un-known mutations. One promising development is COLD-PCR (coamplification at lower denaturation temperature), which enables enrichment of PCR am-plicons containing unknown mutations at any posi-tion, such that they can be subsequently sequenced to identify the exact nucleotide change. SUMMARY: This review summarizes technologies avail-able for detectingminority DNAmutations, placing an emphasis on newer methods that facilitate the enrich-ment of unknown low-levelDNAvariants such that the mutation can subsequently be sequenced. The enrich-ment of minority alleles is imperative in clinical and diagnostic applications, especially in those related to cancer detection, and continued technology develop-ment is warranted