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How to bring light to PCR: TaqMan probe or SYBR Green dye?

Bram De Craene - Apr 24, 2018

As a qPCR trainer, I often get the question why to worry about careful design and validation of dye-based assays when an easier alternative is available: TaqMan probes. This question results from the widespread believe that probe-based assays are superior in specificity to assays that rely on SYBR Green I chemistry for detection.

In this blog post, I want to nuance this point of view: dye-based assays are perfect for many real-time qPCR applications. When you understand the true advantages and disadvantages of probe-based assays, you can reduce the cost while keeping the quality of your qPCR experiments high. And, in the end, isn’t that what we all want?

Topics: primer- gene expression- qPCR- Insider- RT-qPCR- sensitivity

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Benchmarking RNA sequencing sensitivity using transcriptome-wide RT-qPCR data

Jan Hellemans - Aug 26, 2014

In the US FDA-led SEQC (aka MAQC-III) study, different sequencing platforms were tested across more than ten sites using well established reference RNA samples with built-in truths in order to assess the discovery and expression profiling performances of platforms and analysis pipelines (Su, Łabaj, Li et al., Nature Biotechnology, 2014). The entire SEQC data set comprises over 100 billion reads (10 Tb) thus providing a unique resource for thorough assessment of RNA-seq performance. Biogazelle co-authored and complemented this study by defining the first human transcriptome using well-established RT-qPCR technology. Using almost 21,000 PrimePCR qPCR assays (jointly developed by Biogazelle and Bio-Rad), the mRNA expression repertoire of the four MAQC samples was established.

Topics: RNA sequencing- RT-qPCR- sensitivity- read depth

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Novel splice junctions identified in RNA sequencing studies: noise or interesting biology?

Jan Hellemans - Apr 23, 2014

What is a gene? Perhaps a simple question, but no clear answer is given by geneticists. Although the concept of a gene has been shown to be much more complex than ‘a DNA sequence transcribed into RNA and translated into a single protein product’, scientists often ignore this complexity. For example, while the majority of human genes show differential splicing, expression analysis is typically performed in function of genes, not transcripts (PubMed for instance has more than 800,000 ‘gene expression’ papers but only around 2,000 ‘transcript expression’ papers). I do believe that most of the authors of these papers are not ignorant of the concept of alternative splicing, but rather that transcripts are harder to study because less is known about specific transcripts functions than about gene functions, and because transcript analysis used to be more challenging from a technological perspective.

Topics: splice variants- RNA sequencing- RT-qPCR

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