![]() In fact, a number of states are drafting or considering legislation outlawing the use of mRNA vaccines in food animals or, at minimum, requiring their labeling on animal products in grocery stores. These misconceptions about mRNA vaccines have recently spilled over into worries about whether their use in agricultural animals could expose people to components of the vaccine within animal products such as meat or milk. While effective vaccines for COVID-19 should have heralded the benefits of mRNA vaccines, fear and misinformation about their supposed dangers circulated at the same time. Proc Natl Acad Sci USA 105, 10513–10518.The following essay is reprinted with permission from The Conversation, an online publication covering the latest research. Circulating microRNAs as stable blood-based markers for cancer detection. Multiplexed target detection using DNA-binding dye chemistry in droplet digital PCR. Absolute quantification by droplet digital PCR versus analog real-time PCR. 2013), making the study of gene expression even simpler and more precise. It even provides the capacity to do simultaneous detection of target and reference using EvaGreen chemistry (McDermott et al. One advantage of the QX200™ ddPCR System is that it is compatible with existing primers, probes, and protocols for gene expression studies. Contamination detection (e.g., Mycoplasma)īefore treatment: Quantitative evaluation of baseline RNAs and gene expression levelsĪfter treatment: Quantitative re-evaluation of RNAs and gene expression levelsĭigital PCR can be used for studies of gene expression both alone and in combination with other techniques.Residual host cell DNA quantification and sizing.Ratio analysis testing for multivalent RNA therapeutics.RNA quantification for analyzing biodistribution.Quantitative evaluation of RNAs and gene expression to identify potential therapeutic targets.While many advancements are being made, the field of RNA therapeutics is still new and working to address challenges to make RNA-based therapies safer and more viable on a larger scale.ĭroplet Digital PCR technology provides precision and accuracy necessary to address common development, production, and testing challenges and allow RNA therapeutic developers to be confident in their products and maintain workflow efficiency.īio-Rad ddPCR technology supports RNA therapeutics across development, production, and testing. Its extreme precision also makes it a method of choice when operating with low amounts of starting material (such as chromatin immunoprecipitation experiments). Due to its great capacity to discriminate alleles, digital PCR can provide more sensitive detection and accurate measurement of methylation events. Unmethylated CpG is converted to UpG by sodium bisulfite, whereas methylated DNA is unaffected. The most common PCR-based method for the quantification of DNA methylation is the use of different primer pairs that are designed to distinguish methylated from unmethylated CpG islands after treatment with sodium bisulfite (Ku et al. PCR is one of several techniques used for studying methylation. Genomic DNA methylation usually occurs at CpG islands in noncoding regions and generally reduces gene expression. Regulation of gene expression at the genomic level is primarily controlled by the methylation of genomic DNA and by DNA-histone interactions, which are affected by histone modifications such as methylation, phosphorylation, and acetylation. This sensitivity is particularly evident in quantification of low-abundance target, and in discrimination of rare alleles against an abundant wild-type background. One of the greatest advantages of digital PCR for gene expression studies is the exquisite sensitivity of quantification it provides. ddPCR technology does not rely on a standard curve and is insensitive to the number of amplification cycles. Application of Poisson statistical analysis yields absolute quantification. After cycling, the level of fluorescence is measured in each droplet droplets with fluorescence are scored as positive, and those with little or no fluorescence are scored as negative. In Bio-Rad's Droplet Digital™ PCR (ddPCR™) System, each sample is partitioned into many droplets prior to amplification. Either one-step or two-step RT-PCR can be used in ddPCR. Digital PCR can increase the detection of rare transcripts and provide easier and more accurate quantitation. Determination of the presence or absence of particular RNA transcripts and their quantification by reverse-transcription PCR (RT-PCR) is now routine in many laboratories. Most analysis of gene expression is focused on quantifying transcription levels. ![]()
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