Real-time PCR, also called qPCR, stands out as a remarkable technique for monitoring the amplification of target DNA in real time. The qPCR array offers a rapid, robust, and susceptible method for profiling gene expression and quantifying copy number variation (CNV) through real-time PCR. For years, qPCR has been a go-to approach for copy number assays. CNVs represent widespread genetic variations, and their link to disease has garnered growing recognition. Operating akin to regular PCR, qPCR incorporates signaling molecules into the master mix, which are then read after each cycle. The resulting data is normalized to a reference sequence found in consistent amounts across all samples, usually a ubiquitously expressed ‘housekeeping’ gene with a single copy.
A suite of qPCR services, encompassing qPCR assays and PCR services, presents invaluable advantages for research endeavors. Ensuring precise quantification of target nucleic acids sheds light on gene expression levels, genetic variations, and copy number qPCR quantification, which is crucial for comprehensive copy number analysis and copy number variation assay. Leveraging advanced methodologies such as digital PCR analysis and qRT-PCR analysis can guarantee unparalleled sensitivity and specificity, facilitating the detection of even the most minute targets and the differentiation of closely related sequences.
Quantification is performed relative to a ‘normal’ sample with a known copy number set as the baseline. The copy number is determined by comparing the results to a standard curve. As is standard for all qPCR assays, samples, standard curves, and normal controls should be run in triplicate. However, despite the widespread use of qPCR over many years, researchers may feel overwhelmed by the multitude of terms, protocols, and kits available on the market.
Must Read: qPCR Expression Analysis: Probing Gene Expression with Quantitative PCR
When crafting a qPCR assay, adhere to these steps:
- Begin by scouring the literature and databases (like www.rtprimerdb.org) to locate pre-existing primers.
- Select a target sequence for your assay.
- Design primers and probes tailored to your chosen target sequence.
- Verify primer specificity to ensure accurate amplification.
- Evaluate the properties of both primers and probes, including melting temperature (Tm), secondary structure, and complementarity.
- Assess the properties of the PCR product.
- Validate the primers and probes through experimentation, optimizing the protocol for optimal performance.
Consider the following guidelines for optimizing accuracy in qPCR;
- Contamination Risks: Due to its high sensitivity, qPCR is susceptible to contamination, which can significantly bias results. Adhering to the three-room rule during qPCR is crucial to mitigate this risk.
- Even minor errors or deviations in the protocol can be significantly amplified during qPCR: It is crucial to calibrate pipettes regularly and use them accurately, particularly for low volumes. Whenever feasible, utilizing a pre-prepared qPCR mix is recommended to ensure optimal distribution of reagents among samples.
- Maintaining primer integrity and quality is essential for generating high-quality data via qPCR: Storing primers in the appropriate buffer is crucial to ensure their stability. If using DEPC-treated water, which may be slightly acidic, it’s advisable to neutralize it to maximize primer stability.
- Optimizing the qPCR reaction is essential for each new target: Assessing primer efficiency with every new set is crucial. This optimization involves PCR parameters such as primer design and reverse transcription, cycling parameters, and template quantity and quality.
- Successful quantitation depends on determining the optimal RNA quantity: This amount depends on the target’s concentration per sample and may range from picogram to nanogram concentration.
- Comparison between one-step and two-step reverse transcriptase qPCR: One-step RT-qPCR involves conducting the entire process sequentially within a single tube containing the RNA template. This method proves advantageous when working with limited RNA samples, offering heightened sensitivity compared to two-step RT-qPCR while also being less complex.
Streamlined processes guarantee swift turnaround times, empowering you to access dependable data promptly and facilitating informed decision-making across various research domains, including toxicity studies and pharmacokinetic studies. Furthermore, a tailored approach to service provision, including qPCR expression analysis and PCR service, ensures that specific research requirements are met with precision and efficacy, delivering pertinent and actionable outcomes.
