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It has been reported that human mRNA is present in the patient's feces and can be used as a potential marker for the pathological state of the patient's digestive tract. However, the detection of RNA with feces as a sample must overcome two major challenges: First, a large amount of PCR inhibitor in the stool will seriously affect the PCR efficiency and even PCR failure; Second, the majority of the RNA in the stool sample is microbial-derived, human mRNA content. Very few. So this application requires ultra-sensitive nucleic acid separation detection technology.
In this paper, digital PCR was used to analyze the expression levels of up to 70 genes in a large number of frozen fecal samples. GAPDH was used as an internal reference, the probe was labeled with VIC, and each gene was labeled with FAM. All 70 samples have been tested by Shuangtang absorption test, and randomly selected samples were detected by digital PCR and RT-qPCR. The results showed that digital PCR has higher sensitivity than qPCR. Digital PCR-based stool sample RNA detection means can be used to screen nucleic acid markers associated with digestive tract diseases.
It is known that quantitative PCR is obtained by analyzing and processing a real-time fluorescent signal to obtain a so-called Cq value, and then quantifying the sample according to Cq. The Cq value is susceptible to PCR efficiency, especially PCR inhibitors, so qPCR is less sensitive to sensitivity and accuracy when detecting samples with low target nucleic acid abundance and high inhibitory components. Digital PCR, by means of PCR endpoints, determines the way in which the droplets are positive and then quantifies the sample. It is very little affected by PCR efficiency and inhibitors, and is particularly suitable for things like:
1. Detection of pathogenic microorganisms and detection of tumor markers using feces and blood as samples;
2. Environmental biology research and pathogenic microorganism monitoring using soil, sewage, silt, and lees as samples;
3. Identification and content analysis of genetically modified components, animal and plant components, and detection of pathogenic microorganisms (CIQ/CDC) in food materials containing a large number of inhibitors produced by complex processes;
4, with tibia, hair, secretions and excretions, or oral detachment from cells, animal scales and skin, old specimens for the application of forensic medicine, animal genetic diversity management and wildlife protection involving nucleic acid detection and analysis.
[Agapova S, Stephenson K, Manary M, Weisz A, Tarr PI, Mkakosya R, Maleta K, Shulman RJ, Manary M, Shaikn N. Detection of Low Concentration Host mRNA Transcripts in Malawian Children at Risk for Environmental Enteropathy. J Pediatr Gastroenterol Nutr.2012.
In recent years, research on long noncoding RNA (IncRNA) has become a hot topic in the research of small RNA. IncRNA is a general term for a class of RNA molecules with a transcript of more than 200 nucleotides in length. The expression level of IncRNA is generally low relative to the gene encoding the protein. It is important that many of these IncRNAs are not directly involved in gene coding and protein synthesis, but have important functions in post-transcriptional regulation, cleavage and modification of genes, in many life. The activities play an important role in the activities, and have a close relationship with the occurrence, development, diagnosis and treatment of diseases, and quickly become one of the most popular frontier research fields in molecular biology. In addition, the subcellular location of IncRNA is also diverse, distributed in the nucleus, cytoplasm and organelles, and even some of the IncRNA has a unique subcellular location, which may be a new subcellular composition.
Although little research has been done, people have gradually realized that IncRNA has important functions. Therefore, the abnormality of IncRNA is closely related to diseases, and it will become a new research direction for understanding diseases, searching for molecular markers of diseases, and drug targets.
Recently, American scientists published the first paper on the application of digital PCR to IncRNA research at NUCLEC ACID THERAPEUTICS. The article directly quantifies the IncRNA molecule in an absolute quantitative way to determine whether its regulation of gene expression is cis or trans. It also analyzes the expression of different IncRNA and housekeeping genes at the subcellular level.
[David W, Dodd, Keith T. Gagnon, and David R. Corey. Digital Quantitation of Potential Therapeutic Target RNAs. NUCLEIC ACID THERAPEUTICS, Volume 23, Number 2, 2013.]
Researchers from the Fred Hutchinson Cancer Research Center have published in the Nature Method that digital PCR technology can be used to quantify accurate and reproducible plasma and serum microRNA (miRNA) in different situations, which will be miRNA and The use of other nucleic acids for the detection of circulating biomarkers paved the way.
The first author of the article, Dr. Muneesh Tewari, Department of Human Biology, Fred Hutchinson Cancer Research Center, said, "In the field of miRNA diagnostics for circulatory systems, digital PCR can help us conduct biomarker research, directly comparing multiple experiments across the same laboratory, even Test results between different laboratories"
Real-time quantitative PCR (qPCR) has been widely used in analytical testing of blood sample miRNAs. But researchers have found that miRNA qPCR measurements in serum or plasma are highly variable and cannot be used as a stable test for blood biomarkers, so researchers in this field have been looking for a way to be more reliable. A new method of results.
Digital PCR has many advantages over quantitative PCR, such as the absence of a standard curve and the fact that it is not affected by changes in PCR amplification efficiency during different samples and experiments, and absolute quantification can be obtained.
To evaluate the steps of each workflow, including serial dilution preparation, reverse transcription (RT) and PCR amplification, Dr. Tewari and his team performed nested analysis of quantitative PCR by digital PCR on three separate working days. The cDNAs of different dilutions of different synthetic miRNAs in water and plasma were analyzed. It was found that compared with qPCR, digital PCR has higher accuracy in PCR-specific changes (48-72% less variation). coefficient).
Next, the team conducted a side-by-side comparison of qPCR and digital PCR of serum miRNAs. They collected 20 patients with advanced prostate cancer and 20 age-matched male control serum samples for analysis of miR-141 abundance. miR-141 is a proven biomarker for advanced prostate cancer. The researchers prepared different dilutions of qPCR and digital PCR analysis in different three days and tested them. It was found that digital PCR can increase the repeat rate of the laboratory by 7 times compared with qPCR. It was also confirmed in the control study that the results of the digital PCR were more reliable (p=0.0036 vs. p=0.1199).
[Christopher M Hindson, John R Chevillet, Hilary A Briggs, Emily N Gallichotte, Ingrid K Ruf, Benjamin J Hindson, Robert L Vessella & Muneesh Tewari Absolut Quantification by droplet digital PCR versus Analog real-time PCR. Nature Methods | ADVANCE ONLINE PUBLICATION Doi:10.1038/nmeth.2633.]
Recent scientists from the Mount Sinai School of Medicine in New York, USA, have successively published the latest research results of digital PCR applied to embryonic stem cell gene expression research. Embryonic stem cells (ESCs) are referred to as ES cells. ES cells have cell pluripotency and can participate in the developmental potential of various tissues including the gonads under the condition of de-inhibition of differentiation, that is, ES cells have the ability to develop into a complete animal body, which can be used for genetic manipulation and cell differentiation of cells. Provide a wealth of experimental materials.
The researchers found that MicroRNA-137 (miR-137) plays an important role in the differentiation of ES cells into neural cells. Scientists used digital PCR and qPCR to simultaneously verify the expression level of miR-137 under different cell cycle conditions, and found that miR-137 was significantly up-regulated in the metaphase of cells. Combined with other experimental evidence, miR-137 can regulate the proliferation and differentiation of ES cells by inhibiting the expression of downstream transcriptional regulators.
[Ke Jiang, Chunyan Ren, Venugopalan D. Nair MicroRNA-137 Represses Klf4 and Tbx3 During Differentiation of Mouse Embryonic Stem Cells. Stem Cell Research (2013) 11,1299-1313.]
Literature Sharing | Application of Nacia Digital PCR in Gene Expression Analysis
Researchers at the University of Washington School of Medicine and the Pediatrics School of Medicine at the University of California, San Diego have established a robust and reliable RNA-based magnetic bead extraction and digital PCR method for detecting human mRNA associated with EE (tropical bowel disease) from fecal samples. The sensitivity is up to 20 copies of GAPDH mRNA/200mg stool sample.
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