6 Tips to Improve qPCR (part 1 of 3)

qPCR array provides a quick, powerful and sensitive approach for gene expression profiling. To help our customers acquire accurate and consistent results, we are happy to share some tips for better qPCR.

Tip #1. Good primer design is the key to a successful qPCR array.

The success of a qPCR array depends on primer specificity and efficiency, so good primer design is extremely important.  The general rules of primer design for traditional PCR also apply for qPCR arrays: 40-60% GC content; 3′-end GC clamps; no repetitive sequence; no self-dimer, cross-dimer, or hairpin formation; and no long stretches of polypurines or polypyrimidines. Furthermore, there are additional aspects to consider when designing primers for qPCR arrays. First, the amplicon length should be around 100-200bp.  If the amplicon length is too short, the PCR product size will be too similar to the primer dimer to distinguish them from one another without sequencing.  If it is too long, the PCR efficiency will decrease requiring more time for elongation and a great probability of non-specific amplification. Second, many genes are analyzed in qPCR array at the same time under the same PCR conditions, so all primers in the array should have similar optimal annealing temperatures.  Third, many genes have multiple transcript variants due to alternative splicing, therefore, it is best to select the amplicon located in a region shared by all variants. ScienCell uses these criteria to design each and every primer. To save you time on primer design and verification, simply order from our pre-designed GeneQuery™ qPCR array kits, or our primer sets for individual gene analysis.

Tip #2. Closely monitor qPCR array for possible contamination.

Due to the high sensitivity of the qPCR method, contamination may also be amplified exponentially and interfere with accuracy and precision, especially for genes with a low copy number. There are two main sources of contamination: internal contamination from genomic DNA (gDNA) and/or external contamination introduced in sample handling.

To eliminate gDNA contamination from cDNA, the most commonly used method is to treat total RNA samples with DNaseI, followed by deactivation of DNaseI to prevent degradation of the qPCR products. In addition, we recommend using a gDNA control (GDC) to monitor the gDNA contamination in the sample preparation. A primer set targeting a non-transcribed region of the genome can be used for the GDC. In ScienCell’s GeneQuery™ qPCR array kits, a GDC control is included in every plate.

External contamination is usually inadvertently introduced by a DNA containing amplicon sequence. The source of the contamination may originate from gloves worn when opening a cDNA tube, pipettes used to transfer previous PCR products, or even DNA-containing aerosols. To avoid external contamination, the best practice is to have a room or a bench designated as “DNA-free” to handle PCR reagents. Always wear new gloves when working in this area and dedicate a set of pipettes and filter tips for handling “DNA-free” reagents. We recommend spraying DNAZap™ solution from Ambion™ periodically to maintain the lab bench, pipettes, and gloves DNA-free. To monitor external contamination in your qPCR array, include a No Template Control (NTC) containing a primer set that can amplify one housekeeping gene. For the NTC, add all of the reagents (water and polymerase mix) without the template. A negative reading of from this well indicates the array is free of external contamination. In ScienCell’s GeneQuery™ qPCR array kits, a No Template Control (NTC) is also included in every plate to monitor the external contamination.

See 6 Tips to Improve qPCR (part 2 of 3) >>

In our upcoming posts, the following tips will be discussed:

Tip #5. Good Laboratory Practice (GLP) with pipetting helps to improve qPCR accuracy.

Tip #6. Use a separate reverse transcription step to optimize your qRT-PCR

For more information, please visit http://www.sciencellonline.com/products-services/gene-analysis.html, or email info@sciencellonline.com.


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