A universal molecular control for DNA, mRNA and protein expression.
Helen M GunterScott E YoultenAndre L M ReisTim McCubbinBindu Swapna MadalaTed WongIgor StevanovskiArcadi CipponiIra W DevesonNadia S SantiniSarah KummerfeldPeter I CroucherEsteban MarcellinTimothy R MercerPublished in: Nature communications (2024)
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.
Keyphrases
- circulating tumor
- mass spectrometry
- binding protein
- single molecule
- single cell
- cell free
- gene expression
- copy number
- genome wide
- high resolution
- liquid chromatography
- nucleic acid
- amino acid
- protein protein
- dna methylation
- rna seq
- high performance liquid chromatography
- poor prognosis
- circulating tumor cells
- gas chromatography
- induced apoptosis
- label free
- cell proliferation
- cell death
- small molecule
- signaling pathway
- transcription factor
- cell cycle arrest
- microbial community
- ms ms
- real time pcr
- loop mediated isothermal amplification