Real Time qPCR Analysis CRO And Digital ddPCR Assay Services To Accelerate Your Gene Expression, Biodistribution, Viral Shedding, Mutation Detection, Genetic Variation, And Genotyping Studies!
Absolute Quantitation Digital dPCR TaqMan Assay in RNA Isolates from Human Skin
Customized Digital PCR Analysis With Multiple Housekeeping Genes in RNA from NHP Tissues
Digital PCR Service in DNA Isolates from Cell Lines
Digital ddPCR Service With Custom Designed Primers And Probes in DNA Isolates from NHP Tissues
Host residual DNA (hrDNA) Digital PCR Service in Cell Culture Media
Multiplex Reverse-Transcriptase qPCR Expression Analysis in RNA Isolates from Human Skin
QRT PCR Analysis With Multiple Housekeeping Genes in RNA from NHP Tissues
Multiplex Real Time qPCR in DNA Isolates from Cell Lines
20+
Years of experience in designing, optimizing, and validating qPCR assays
400+
Custom Bioanalytical Methods Completed Within Budget
We sincerely collaborate with you to achieve our common bioanalysis goals and speed up your qPCR assay or ddPCR analysis. Generally, we can do qPCR assay development and validate qPCR or ddPCR assays in six-to-eight weeks. Then, we can deliver audited documentation of GLP/MIQE/FFP qualification experiments in a few weeks. We have over 20 years of experience in designing, optimizing, and validating qPCR assays for biotech companies. So, we use our core strengths- operational excellence, regulatory expertise, and scientific experience to ensure your qPCR/ddPCR analysis is cost-effective and resource-efficient.
Our team values clear communication between sponsors and our scientists, project managers, lab staff, etc. Could you please provide us additional introductory and background information about your analyte(s), preferred critical reagents, qPCR assay supplies, previous qPCR analysis data from experimental run(s), study protocols (with # of cohorts, samples), and any crucial ddPCR assay or RT-PCR analysis needs? All this information will help us accurately and efficiently define the scope of work for quotation and timeline estimation.
NorthEast BioLab truly goes that extra mile, and we hope to continue enjoying seminal interactions with them.
NorthEast BioLab presents a science-based, hands-on, no-frills approach on the latest bioanalytical platforms.
NorthEast BioLab is by far the most responsive and thorough bioanalysis lab services CRO.
As a small company, our vendors are key members of our project teams. The scientists at NorthEast BioLab are technical experts, who produce high-quality data, on-time, and on-budget.
We partnered on several programs and found NorthEast BioLab responsive, collaborative, and reliable.
We trust NorthEast BioLab to design and execute the most streamlined and impactful bioanalytical projects.
Discover Digital PCR Analysis And Real-Time Quantitative PCR For Absolute Quantitation Of Genomic Alterations In Copy Number Variation Analysis …
FDA Audited Bioanlaysis Lab qPCR CRO Offering qPCR Assay Development, Qalidation As Well As Agile ddPCR Analysis Services To Biotech Companies
We Judiciously Invest In Our People, Solutions And Infrastructure, And Regularly Review Our Business Processes And Practices To Exceed Sponsor Expectations
Answers To Additional qPCR Services Questions Popular Among Our Potential Sponsors.
PCR stands for Polymerase Chain Reaction. This technique was first developed in the 1980s, and since then, it has been used to generate a large amount of DNA from a small DNA volume.
PCR is a technique in itself. However, researchers often employ PCR in other processes, such as gel electrophoresis, DNA sequencing, and pathogen detection. PCR is fundamentally a diagnostic method used in different domains, including microbiology, virology, mycology, parasitology, and dentistry.
PCR amplifies a specific section of DNA. The process begins with a DNA template. An enzyme, polymerase, copy this piece of DNA. The PCR reaction has a heating and cooling cycle. Researchers employ this cycle to separate the DNA strand and make multiple copies of the same strand. The final assay reaction thus contains a large number of identical DNA copies that researchers can use for further analysis, such as Southern blotting and sequencing.
Quantitative PCR (qPCR) is similar to traditional PCR, but it can quantify the amount of DNA in the sample. qPCR analysis involves probes and fluorescent dyes that bind to the target DNA. These probes, when excited by a light source, emit different wavelength lights.
Researchers measure the wavelength of each emitted light to determine the amount of target DNA present in the sample. Besides, one may monitor DNA amplification in the actual moment by determining the increase in fluorescence in real time.
Reverse-transcriptase PCR (RT-PCR) is a PCR sub-type that generates cDNA from template mRNA molecules. RT-PCR can detect and quantify RNA viruses. The first step in RT-PCR analysis is reverse transcribing the mRNA template using reverse transcriptase enzyme. This cDNA is then amplified using PCR. RT-PCR is a sensitive and accurate technique to detect RNA viruses present at very low levels.
qPCR can measure gene expression on its own. In qPCR expression analysis, a section of DNA is read to synthesize the mRNA, which then produces the protein of interest. However, in RT-PCR, reverse transcriptase creates cDNA, which is then used in PCR to calculate the mRNA transcripts.
Droplet Digital PCR (ddPCR) is an advanced version of qPCR and qRT PCR analysis. ddPCR gene expression can identify and amplify specific gene sequences for subsequent quantification. While qPCR has been a gold standard for evaluating gDNA and cDNA levels, it has limitations when it comes to assessing small differences in gene expression. ddPCR gene expression reduces these limitations by employing a partition in the traditional qPCR analysis. This ddPCR method divides a typical qPCR run into thousands of droplets. Each droplet is in the nanoliter range. Hence, ddPCR gene expression determines the target molecules based on Poisson Distribution. Such accurate analysis has made ddPCR gene expression the primary technique in the absolute quantitation of copy number variation and rare mutations.
Digital PCR (dPCR) assay is a novel technique for measuring and identifying nucleic acids that can quantify them precisely and detect rare variants. It works by dividing and isolating a DNA or RNA sample into many separate, parallel PCR reactions; some of these reactions have the target molecule (positive) and others do not (negative). Here, a single DNA or RNA molecule can be amplified by a factor of a million or more. During amplification, TaqMan® Assays with dye-labeled probes bind to specific sequences to identify targets. When the target sequence is absent, no signal is produced. After PCR analysis, the proportion of negative reactions is used to calculate the exact number of target molecules in the sample, without requiring standards or internal controls.
Digital PCR analysis has many uses and is getting increasingly deployed to:
Some differentiating features of the QuantStudio™ Absolute Q™ Digital PCR dPCR System include:
The primary difference between the two methods is that qPCR measures in real-time. This real-time feature means researchers can monitor target DNA amplification as it is happening.
Secondly, qPCR employs probes or fluorescent dyes. These probes quantify the total amount of target DNA present in the study sample.
Finally, the main function of PCR analysis is to amplify DNA and help in other downstream applications. On the other hand, qPCR is employed widely to detect and quantify RNA viruses.
In gene expression analysis, qPCR assays can determine the amount of mRNA present in the sample. A small mRNA region is amplified, while the qPCR tool measures the intensity of individual probes. As the number of cycles increases, the fluorescent signals increase with an increase in amplicons.
The Ct value represents the primary qPCR experience. The threshold in qPCR analysis is in the linear phase, and the curve crossing the threshold is the measured Ct value. The threshold value is unique for each qPCR assay.
The primary principle of qPCR analysis is that at each assay cycle, the PCR products double in number. Often, qPCR analysis gives a relative expression, which varies in the gene expression of the assay sample. Researchers may also determine the absolute quantification of a gene. However, absolute quantification requires a standard, which is generally the cloning of cDNA into a vector.
Real Time-PCR/qPCR quantification can be absolute or relative. Absolute quantitation determines unknown samples by interpolating their copy number relative to those obtained from a standard curve. Absolute quantitation may be used to study the correlation between viral or gene copy numbers and disease state. It is important to understand the exact copy number of the target RNA in a biological sample to be able to study the progress of the disease. Relative quantitation is used to analyze how the activity of genes in a sample is increased or decreased relative to a reference, control sample that has a normal or baseline gene activity. Relative quantitation is used for comparisons of healthy and diseased subjects, for determining genetic or phenotype related differences between samples., or to compare disease or disease treatment sample biomarkers to baseline levels. Both methods involve normalization of gene quantitation by simultaneous or parallel measurement of a housekeeping gene (for eg. beta actin or GAPDH) – the expression levels of which are relatively stable and are not altered by disease state or treatment. This ensures that differences in absolute or relative level of the gene changes observed are from disease state or treatment and not from differences in nucleic acid loading.
There are two approaches to qPCR analysis: dye-based and probe-based. Let us dive deep into each of these qPCR types.
This method measures the fluorescent signals, specifically the binding of the dye to the double-stranded DNA. Initially, the dye shows a weak signal. However, after binding to the double-stranded DNA, the signal increases dramatically. Hence, target sequence amplification increases the fluorescence, which directly relates to the amount of double-stranded DNA in the sample. This approach needs only two primers specific to the sequence. Hence, dye-based qPCR is a rapid way to assess a large number of study samples.
However, dye-based methods can detect all double-stranded DNA present in the sample. This non-specific detection can include primer dimers and non-specific products, leading to inaccurate results. Hence, reaction specificity is verified using the denaturing/melting curve after each qPCR run. Such an approach ensures that only the target DNA is amplified. Moreover, another limitation of this approach is that dye-based qPCR can only quantify one target sequence at a time.
Probe-based qPCR measures the fluorescent signal of sequence-specific fluorophore-labeled probes. This method is more specific than a dye-based approach and hence is preferred in diagnostic applications.
Probe-based qPCR is a multiplexed method that can quantify multiple targets in a single sample reaction. This multiplexing approach employs a specific fluorophore for each target. Probe designs vary among experiments. However, hydrolysis probes are most commonly used in probe-based qPCR.
qPCR is a rapid and sensitive tool for the accurate determination of nucleic acids in diverse biological samples. Their applications include cancer phenotyping, gene expression analysis, and the detection of GMOs in food.
In research capacities, qPCR can provide quantitative analysis of gene copy numbers and the presence of mutant genes. Besides, qPCR can be combined with RT-PCR to quantitate alterations in gene expression. For example, researchers can monitor changes in mRNA levels to assess the effect of drugs and environmental conditions on gene expression levels.
