A novel microRNA (miRNA) quantification method has been developed using stem-loop RT followed by TaqMan PCR analysis

A novel microRNA (miRNA) quantification method has been developed using stem-loop RT followed by TaqMan PCR analysis. Stem-loop RT primers are better than conventional ones in terms of RT efficiency and specificity. TaqMan miRNA assays are specific for mature miRNAs and discriminate among related miRNAs that differ by as little as one nucleotide. Furthermore, they are not affected by genomic DNA contamination. Precise quantification is achieved routinely with as little as 25 pg of total RNA for most miRNAs. In fact, the high sensitivity, specificity and precision of this method allows for direct analysis of a single cell without nucleic acid purification. Like standard TaqMan gene expression assays, TaqMan miRNA assays exhibit a dynamic range of seven orders of magnitude. Quantification of five miRNAs in seven mouse tissues showed variation from less than 10 to more than 30,000 copies per cell. This method enables fast, accurate and sensitive miRNA expression profiling and can identify and monitor potential biomarkers specific to tissues or diseases. Stem-loop RT-PCR can be used for the quantification of other small RNA molecules such as short interfering RNAs (siRNAs). Furthermore, the concept of stem-loop RT primer design could be applied in small RNA cloning and multiplex assays for better specificity and efficiency.
A novel microRNA (miRNA) quantification technique has been developed utilizing stem-loop RT adopted by TaqMan PCR evaluation. Stem-loop RT primers are higher than standard ones by way of RT effectivity and specificity. TaqMan miRNA assays are particular for mature miRNAs and discriminate amongst associated miRNAs that differ by as little as one nucleotide. Moreover, they don’t seem to be affected by genomic DNA contamination.
Exact quantification is achieved routinely with as little as 25 pg of complete RNA for many miRNAs. Actually, the excessive sensitivity, specificity and precision of this technique permits for direct evaluation of a single cell with out nucleic acid purification. Like customary TaqMan gene expression assays, TaqMan miRNA assays exhibit a dynamic vary of seven orders of magnitude.
Quantification of 5 miRNAs in seven mouse tissues confirmed variation from lower than 10 to greater than 30,000 copies per cell. This technique permits quick, correct and delicate miRNA expression profiling and might establish and monitor potential biomarkers particular to tissues or illnesses.
Stem-loop RT-PCR can be utilized for the quantification of different small RNA molecules comparable to brief interfering RNAs (siRNAs). Moreover, the idea of stem-loop RT primer design may very well be utilized in small RNA cloning and multiplex assays for higher specificity and effectivity.
A novel microRNA (miRNA) quantification method has been developed using stem-loop RT followed by TaqMan PCR analysis. Stem-loop RT primers are better than conventional ones in terms of RT efficiency and specificity. TaqMan miRNA assays are specific for mature miRNAs and discriminate among related miRNAs that differ by as little as one nucleotide. Furthermore, they are not affected by genomic DNA contamination. Precise quantification is achieved routinely with as little as 25 pg of total RNA for most miRNAs. In fact, the high sensitivity, specificity and precision of this method allows for direct analysis of a single cell without nucleic acid purification. Like standard TaqMan gene expression assays, TaqMan miRNA assays exhibit a dynamic range of seven orders of magnitude. Quantification of five miRNAs in seven mouse tissues showed variation from less than 10 to more than 30,000 copies per cell. This method enables fast, accurate and sensitive miRNA expression profiling and can identify and monitor potential biomarkers specific to tissues or diseases. Stem-loop RT-PCR can be used for the quantification of other small RNA molecules such as short interfering RNAs (siRNAs). Furthermore, the concept of stem-loop RT primer design could be applied in small RNA cloning and multiplex assays for better specificity and efficiency.

Difficult the “gold customary” of colony-forming models – Validation of a multiplex real-time PCR for quantification of viable Campylobacter spp. in meat rinses

Campylobacter jejuni is the main bacterial food-borne pathogen in Europe. Regardless of the accepted limits of cultural detection of the fastidious bacterium, the “gold customary” in meals microbiology continues to be the willpower of colony-forming models (CFU). In its place, a stay/useless differentiating qPCR has been established, utilizing propidium monoazide (PMA) as DNA-intercalating crosslink agent for inactivating DNA from useless, membrane-compromised cells.
The PMA remedy was mixed with the addition of an inner pattern course of management (ISPC), i.e. a recognized variety of useless C. sputorum cells to the samples. The ISPC permits i), monitoring the efficient discount of useless cell sign by the light-activated DNA-intercalating dye PMA, and ii), compensation for potential DNA losses throughout processing.
Right here, we optimized the tactic for routine utility and carried out a full validation of the tactic in response to ISO 16140-2:2016(E) for the quantification of stay thermophilic Campylobacter spp. in meat rinses towards the classical enumeration technique ISO 10272-2:2017.
As a way to render the tactic relevant and cost-effective for sensible utility, the ISPC was lyophilized to be distributable to routine laboratories. As well as, a triplex qPCR was established to concurrently quantify thermophilic Campylobacter, the ISPC and an inner amplification management (IAC). Its efficiency was statistically just like the 2 duplex qPCRs as much as a contamination degree of 4.7 log10Campylobacter per ml of meat rinse.
The restrict of quantification (LOQ) of the choice technique was round 20 genomic equivalents per PCR response, i.e. 2.three log10 stay Campylobacter per ml of pattern. The choice technique handed a relative trueness research, confirming the robustness towards completely different meat rinses, and displayed adequate accuracy inside the limits set in ISO 16140-2:2016(E).
Lastly, the tactic was validated in an interlaboratory ring trial, confirming that the choice technique was match for goal with a bent of improved repeatability and reproducibility in comparison with the reference technique for CFU willpower. Campylobacter served as a mannequin organism, difficult CFU as “gold customary” and will assist in steering to the final acceptance of stay/useless differentiating qPCR strategies for the detection of food-borne pathogens.

Analysis of strategies for plant genomic DNA sequence evaluation with out DNA and PCR product purification

Genome-editing applied sciences are broadly used to characterize gene features and enhance the options of agricultural vegetation. Though sequence evaluation of gene enhancing goal DNA is probably the most dependable technique of screening gene-edited vegetation, the present DNA sequence evaluation strategies are time consuming and labor intensive as a result of they embody genomic DNA and polymerase chain response (PCR) product purification.
On this research, seven strategies had been carried out for sequence evaluation of plant genomic DNA with and/or with out genomic DNA and PCR product purification. Consequently, good-quality sequencing chromatograms had been obtained utilizing all strategies.
Outcomes confirmed that the partial genomic DNA sequence of Nicotiana benthamiana and Arabidopsis thaliana may very well be sufficiently analyzed with out plant genomic DNA and PCR product purification. Moreover, screening of gene-edited N. benthamiana was profitable utilizing the current strategies. Due to this fact, the examined strategies might cut back the time, simplify the workflow of plant gene evaluation, and assist in screening gene-edited vegetation.

Close to real-time willpower of B.1.1.7 in proportion to complete SARS-CoV-2 viral load in wastewater utilizing an allele-specific primer extension PCR technique

The coronavirus illness 2019 (COVID-19) pandemic attributable to the extreme acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has claimed tens of millions of lives thus far. Antigenic drift has resulted in viral variants with putatively larger transmissibility, virulence, or each.
Early and close to real-time detection of those variants of concern (VOC) and the flexibility to precisely comply with their incidence and prevalence in communities is wanting. Wastewater-based epidemiology (WBE), which makes use of nucleic acid amplification exams to detect viral fragments, is a dependable proxy of COVID-19 incidence and prevalence, and thus gives the potential to observe VOC viral load in a given inhabitants.
Right here, we describe and validate a primer extension PCR technique concentrating on a signature mutation within the N gene of SARS-CoV-2. This permits quantification of B.1.1.7 versus non-B.1.1.7 allele frequency in wastewater with out the necessity to make use of quantitative RT-PCR customary curves.
We present that the wastewater B.1.1.7 profile correlates with its scientific counterpart and advantages from a close to real-time and facile information assortment and reporting pipeline. This assay may be shortly carried out inside a present SARS-CoV-2 WBE framework with minimal value; permitting early and contemporaneous estimates of B.1.1.7 neighborhood transmission previous to, or in lieu of, scientific screening and identification.
[Linking template=”default” type=”products” search=”Anti-Human Relaxin-3″ header=”3″ limit=”144″ start=”4″ showCatalogNumber=”true” showSize=”true” showSupplier=”true” showPrice=”true” showDescription=”true” showAdditionalInformation=”true” showImage=”true” showSchemaMarkup=”true” imageWidth=”” imageHeight=””]
Our research demonstrates that this technique can present public well being models with an extra and far wanted device to quickly triangulate VOC incidence/prevalence with excessive sensitivity and lineage specificity.

Leave a Reply

Your email address will not be published. Required fields are marked *

Related Post