When you are optimizing a new experiment and you need to test many primer pairs, it is usually best to start with SYBR Green. SYBR Green enables you to find the most specific primers without the need to design and purchase expensive probes. SYBR Green also allows melting curve analysis, which can reveal undesirable primer-dimers or other nonspecific PCR products. The use of Gene Scientific’s SYBR Green qPCR Master Mixes together with our AccuAmp qPCR Primer Assays produces guaranteed performance of high specificity and amplification efficiency.

Absolute quantification is a method in which unknown samples are quantified by comparing them to a standard curve. The reliability of this method is dependent on identical amplification efficiencies between the samples compared. Relative quantification is the more commonly used method for gene expression studies and is expressed as the fold difference of gene expression between a test and a control.

In relative quantification, reference genes are used to normalize against variation in sample quality and quantity. A good reference is not up or down regulated by the effect that needs studying, (e.g. condition, drug, therapy, medium etc.). The number of reference genes needed depends on the assay, the accuracy needed and the references chosen. Several reference gene candidates should be tested to find the most suitable combination. You may also review literatures in your field to determine which reference gene(s) other researchers commonly use.

ROX and Fluorescein are passive reference dyes that are used to normalize against fluorescence variation between wells or experiments caused by pipetting/instrument variability. They do not take part in the PCR reaction and their fluorescences remain constant during the PCR reaction.

Poor quality and/or concentration of qPCR templates, primers, or reagents may lead to qPCR failures. Please include appropriate controls to eliminate these possibilities, and make sure your genes are expressed in your samples and are represented in the cDNA preparation. It is also important to activate hot-start Taq DNA polymerase at the start of the cycling program (10 min at 95°C), and use optical caps or sealing on tubes or plates.

The reaction may be contaminated with DNA template or PCR product from a previous PCR, or there exist primer dimers. Be sure to set up qPCR reaction in a clean, contamination-free workspace, and use validated primers.

AccuAmp qPCR Master Mixes increase amplification specificity/efficiency, and include specially designed and optimized buffer that is desirable for different templates with varying GC content.

Hot start Taq polymerase is used to improve the sensitivity and specificity of PCR amplifications by avoiding amplification of non-specific products and primer dimmers during the initial reaction setup. The hot start Taq polymerase is inactive at room temperature, and is activated after heating at 95°C for 10 minutes.

Designing primers crossing intron-exon junction may avoid amplification from the contaminating genomic DNA in some cases. However, the method may not be helpful if the cDNA is from a single-exon gene, or there exist processed pseudogenes, which are genomic DNA sequences similar to normal genes/mRNA sequences but non-functional. The number of identified human pseudogenes is around 20,000. Therefore, it is essential that the starting RNA sample is free of genomic DNA to ensure accuracy of real-time RT-PCR results.

The PCR amplification efficiency tends to decrease as the amplicon size increases. Targets should ideally be 60-200 bp in length.

Yes. We recommend reactions to be mixed by pipetting or gentle vortexing followed by brief spinning to eliminate any bubbles that can interfere with the optical detection.