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A universal molecular control for DNA, mRNA and protein expression.

ABSTRACT: The expression of genes encompasses their transcription into mRNA followed by translation into protein. In recent years, next-generation sequencing and mass spectrometry methods have profiled DNA, RNA and protein abundance in cells. However, there are currently no reference standards that are compatible across these genomic, transcriptomic and proteomic methods, and provide an integrated measure of gene expression. Here, we use synthetic biology principles to engineer a multi-omics control, termed pREF, that can act as a universal molecular standard for next-generation sequencing and mass spectrometry methods. The pREF sequence encodes 21 synthetic genes that can be in vitro transcribed into spike-in mRNA controls, and in vitro translated to generate matched protein controls. The synthetic genes provide qualitative controls that can measure sensitivity and quantitative accuracy of DNA, RNA and peptide detection. We demonstrate the use of pREF in metagenome DNA sequencing and RNA sequencing experiments and evaluate the quantification of proteins using mass spectrometry. Unlike previous spike-in controls, pREF can be independently propagated and the synthetic mRNA and protein controls can be sustainably prepared by recipient laboratories using common molecular biology techniques. Together, this provides a universal synthetic standard able to integrate genomic, transcriptomic and proteomic methods.

SUBMITTER: Gunter HM

PROVIDER: S-EPMC10954659 | biostudies-literature | 2024 Mar

REPOSITORIES: biostudies-literature

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A universal molecular control for DNA, mRNA and protein expression.

Gunter Helen M HM Youlten Scott E SE Reis Andre L M ALM McCubbin Tim T Madala Bindu Swapna BS Wong Ted T Stevanovski Igor I Cipponi Arcadi A Deveson Ira W IW Santini Nadia S NS Kummerfeld Sarah S Croucher Peter I PI Marcellin Esteban E Mercer Tim R TR

Nature communications 20240320 1

The expression of genes encompasses their transcription into mRNA followed by translation into protein. In recent years, next-generation sequencing and mass spectrometry methods have profiled DNA, RNA and protein abundance in cells. However, there are currently no reference standards that are compatible across these genomic, transcriptomic and proteomic methods, and provide an integrated measure of gene expression. Here, we use synthetic biology principles to engineer a multi-omics control, term ...[more]

PMID: 38509097

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Project description:BackgroundPhytoplasma are obligate intracellular plant-pathogenic bacteria that infect a broad range of plant species and are transmitted by different insect species. Quantitative real-time PCR (qPCR) is one of the most commonly used techniques for pathogen detection, especially for pathogens that cannot be cultivated outside their host like phytoplasma. PCR analysis requires the purification of total DNA from the sample and subsequent amplification of pathogen DNA with specific primers. The purified DNA contains mainly host DNA and only a marginal proportion is of phytoplasmal origin. Therefore, detection of phytoplasma DNA in a host DNA background must be sensitive, specific and reliable and is highly dependent on the quality and concentration of the purified DNA. DNA quality and concentration and the presence of PCR-inhibitors therefore have a direct impact on pathogen detection. Thus, it is indispensable for PCR-based diagnostic tests to validate the DNA preparation and DNA integrity before interpreting diagnostic results, especially in case that no pathogen DNA is detected. The use of an internal control allows to evaluate DNA integrity and the detection of PCR-inhibiting substances. Internal controls are generally host-specific or limited to a defined group of related species. A control suitable for the broad range of phytoplasma hosts comprising different insect and plant species is still missing.ResultsWe developed a primer and probe combination that allows amplification of a conserved stretch of the eukaryotic 28S rDNA gene. The developed endogenous qPCR control serves as a DNA quality control and allows the analysis of different eukaryotic host species, including plants, insects, fish, fungi, mammals and human with a single primer/probe set in single- or multiplex assays.ConclusionsQuality and performance control is indispensable for pathogen detection by qPCR. Several plant pathogens are transmitted by insects and have a broad range of host species. The newly developed endogenous control can be used with all so far tested eukaryotic species and since multiplexing is possible, the described primer and probe set can be easily combined with other PCR-based pathogen detection systems.

Project description:Previous studies in yeast have supported the view that post-transcriptional regulation of protein abundances may be more important than previously believed. Here we ask the question: "In a physiological regulatory process (the response of mammalian kidney cells to the hormone vasopressin), what fraction of the expressed proteome undergoes a change in abundance and what fraction of the regulated proteins have corresponding changes in mRNA levels?" In humans and other mammals, vasopressin fulfills a vital homeostatic role (viz. regulation of renal water excretion) by regulating the water channel aquaporin-2 in collecting duct cells. To address the question posed, we utilized large-scale quantitative protein mass spectrometry (LC-MS/MS) employing stable isotopic labeling in cultured mpkCCD cells ('SILAC') coupled with transcriptomic profiling using oligonucleotide expression arrays (Affymetrix). Preliminary studies analyzing two nominally identical control samples by SILAC LC-MS/MS yielded a relative S.D. of 13% (for ratios), establishing the precision of the SILAC approach in our hands. We quantified nearly 3000 proteins with nontargeted SILAC LC-MS/MS, comparing vasopressin- versus vehicle-treated samples. Of these proteins 786 of them were quantified in each of 3 experiments, allowing statistical analysis and 188 of these showed significant vasopressin-induced changes in abundance, including aquaporin-2 (20-fold increase). Among the proteins with statistically significant abundance changes, a large fraction (at least one-third) was found to lack changes in the corresponding mRNA species (despite sufficient statistical power), indicating that post-transcriptional regulation of protein abundance plays an important role in the vasopressin response. Bioinformatic analysis of the regulated proteins (versus all transcripts) shows enrichment of glutathione S-transferase isoforms as well as proteins involved in organization of the actin cytoskeleton. The latter suggests that long-term regulatory processes may contribute to actomyosin-dependent trafficking of the water channel aquaporin-2. The results provide impetus for increased focus on translational regulation and regulation of protein degradation in physiological control in mammalian epithelial cells.

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A universal molecular control for DNA, mRNA and protein expression.

Publications

A universal molecular control for DNA, mRNA and protein expression.

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