SPUD: A quantitative PCR assay for the detection of inhibitors in nucleic acid preparations

Nolan, Tania; Hands, Rebecca; Ogunkolade, W; Bustin, Stephen A.

doi:10.1016/j.ab.2006.01.051

Cited by 218 publications

(168 citation statements)

References 17 publications

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“…The presence of inhibitory factors in the RNA samples was tested using the SPUD assay (Nolan et al 2006). Two micrograms of total RNA were reverse transcribed into cDNA using Superscript III reverse transcriptase and anchored oligo(dT) 20 primers (Invitrogen).…”

Section: Methodsmentioning

confidence: 99%

The Role of MITF Phosphorylation Sites During Coat Color and Eye Development in Mice Analyzed by Bacterial Artificial Chromosome Transgene Rescue

et al. 2009

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The microphthalmia-associated transcription factor (Mitf) has emerged as an important model for gene regulation in eukaryotic organisms. In vertebrates, it regulates the development of several cell types including melanocytes and has also been shown to play an important role in melanoma. In vitro, the activity of MITF is regulated by multiple signaling pathways, including the KITL/KIT/B-Raf pathway, which results in phosphorylation of MITF on serine residues 73 and 409. However, the precise role of signaling to MITF in vivo remains largely unknown. Here, we use a BAC transgene rescue approach to introduce specific mutations in MITF to study the importance of specific phospho-acceptor sites and protein domains. We show that mice that carry a BAC transgene where single-amino-acid substitutions have been made in the Mitf gene rescue the phenotype of the loss-of-function mutations in Mitf. This may indicate that signaling from KIT to MITF affects other phospho-acceptor sites in MITF or that alternative sites can be phosphorylated when Ser73 and Ser409 have been mutated. Our results have implications for understanding signaling to transcription factors. Furthermore, as MITF and signaling mechanisms have been shown to play an important role in melanomas, our findings may lead to novel insights into this resilient disease.

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Section: Methodsmentioning

confidence: 99%

The Role of MITF Phosphorylation Sites During Coat Color and Eye Development in Mice Analyzed by Bacterial Artificial Chromosome Transgene Rescue

et al. 2009

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“…Purity does not refer to a sample's A 260 / A 280 ratio, but rather encompasses the absence of inhibitors of either the RT or the PCR reaction. Inhibition is a well-known yet poorly described phenomenon and we were the fi rst to propose a universal method for inhibition testing that involves the use of a template expressed only in potatoes [ 16 ]. The technique, called SPUD, compares the C q s obtained from the amplifi cation of SPUD templates suspended in water with those obtained from SPUD templates spiked into sample preparations.…”

Section: Why the Need For Such Detail?mentioning

confidence: 99%

Minimum Information Necessary for Quantitative Real-Time PCR Experiments

Johnson

Nour

Nolan³

et al. 2014

Methods in Molecular Biology

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The MIQE (minimum information for the publication of quantitative real-time PCR) guidelines were published in 2009 with the twin aims of providing a blueprint for good real-time quantitative polymerase chain reaction (qPCR) assay design and encouraging the comprehensive reporting of qPCR protocols. It had become increasingly clear that variable pre-assay conditions, poor assay design, and incorrect data analysis were leading to the routine publication of data that were often inconsistent, inaccurate, and wrong. The problem was exacerbated by a lack of transparency of reporting, with the details of technical information inadequate for the purpose of assessing the validity of published qPCR data. This had, and continues to have serious implications for basic research, reducing the potential for translating fi ndings into valuable applications and potentially devastating consequences for clinical practice. Today, the rationale underlying the MIQE guidelines has become widely accepted, with more than 2,200 citations by March 2014 and editorials in Nature and related publications acknowledging the enormity of the problem. However, the problem we now face is rather serious: thousands of publications that report suspect data are populating and corrupting the peer-reviewed scientifi c literature. It will be some time before the many contradictions apparent in every area of the life sciences are corrected.

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“…Because quality of the template DNA is one of the most important determinants of the sensitivity, accuracy and reliability of any PCR [75], care should be taken to avoid the presence of inhibitory compounds in the extracted DNA solution. Three potential mechanisms could inhibit PCRs: binding of the inhibitor to the polymerase, interaction of the inhibitor with the DNA, and interaction with the polymerase during primer extension.…”

Section: Inhibition Testmentioning

confidence: 99%

Determination of Denitrification Genes Abundance in Environmental Samples

Correa‐Galeote¹,

Tortosa²,

Bedmar³

2013

Metagenomics

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Diversity of microorganisms involved in the biogeochemical N cycle is of fundamental interest in microbial ecology. Denitrification is a key step in the cycle by which nitrate is reduced to dinitrogen gas via the soluble nitrite and the gaseous compounds nitric oxide and nitrous oxide. The process is carried out by the sequential activity of the nitrate, nitrite, nitric oxide, and nitrous oxide reductase enzyme, respectively. The fluorescence-based quantitative real-time polymerase chain reaction (qPCR) is widely used for quantification of nucleic acids in samples obtained from numerous, diverse sources. Here, we provide a well-proven methodology for isolation of DNA from environmental samples and describe relevant experimental conditions for utilization of qPCR to assay the 16S rRNA and nar/nap, nirK/nirS, c-nor/qnor, and nos denitrification genes that encode synthesis of denitrifying enzymes. The ISO 11063 standard method and MIQUE guidelines are considered with the aim to increase experimental transparency.

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SPUD: A quantitative PCR assay for the detection of inhibitors in nucleic acid preparations

Cited by 218 publications

References 17 publications

The Role of MITF Phosphorylation Sites During Coat Color and Eye Development in Mice Analyzed by Bacterial Artificial Chromosome Transgene Rescue

The Role of MITF Phosphorylation Sites During Coat Color and Eye Development in Mice Analyzed by Bacterial Artificial Chromosome Transgene Rescue

Minimum Information Necessary for Quantitative Real-Time PCR Experiments

Determination of Denitrification Genes Abundance in Environmental Samples

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