Successful qPCR depends on various things. Most scientists have had a hard time with PCR amplification for some time. Quantitative PCR, or qPCR, is an essential tool. However, like traditional PCR, qPCR can be frustrating. However, several things can help achieve success, depending on the design of the experiment. Errors can lead to non-specific products or low yields. The following are ways you can enhance your qPCR efficiency for better results.

Use High-Quality RNA

Impure or low-quality RNA can lower the efficiency of your RT reaction and reduce the yields. Preparing the RNA from fresh tissue or tissue that has passed through treatment with an RNA stabilization solution is crucial. If you cannot find an utterly intact RNA, you can design primers that will strengthen the internal part of the gene. Note that an RNA that has been partially degraded may not give accurate results or gene expression when seeking quantitative RT-PCR.

Choose a Good Probe and Primer Design 

For efficient primer and QPCR probes, it may be advisable to use primer design software. This helps adjust the parameters for optimal probe and primer design. The parameters consider essential factors like amplicon size, complementarity, and secondary structure. It is also recommended to restrict the number of identical nucleotide runs. It can also be a good idea to use a predesigned primer instead of going through the entire process of designing. You can buy an assay with everything you need, from optimized sequence-specific probes to target primers, without design optimization or lengthy melt-curve analysis.

Prevent Cross-Contamination

It is crucial to clean all the PCR surfaces to avoid cross-contamination. You can use a DNA decontamination substance that destroys all contaminants. Use a No Template Control (NTC) across to remove contamination on surfaces and reagents. Do not synthesize any product in the NTC. If you amplify it, the amplicon has contaminated the RT-PCR reagents.

Set the Baseline and Threshold Correctly

The best way to get the correct Ct values is to set the baseline two cycles before the Ct value of the abundant sample. The threshold should also be selected during the exponential phase of the product to get real-time PCR data.

Use the Right Curve Range 

You must prepare standard curves for each gene of interest to quantitate RNA or verify reaction efficiencies for comparative quantitation. Ensure the curve extends below and above the expected abundance of the target.

Check Optimization

There is no shortcut to optimizing your PCR amplification than optimization. Optimization factors include primer design, magnesium concentration, template quality, buffer composition, enzyme concentration, cycling parameters, and PCR enhancers.

Decide Type of Results

Deciding the type of results you aim to get is also essential. QPCR allows you to determine the absolute or relative quantitation of the target. It is crucial to understand that there is a difference between the two. Absolute quantitation requires creating a standard curve with templates of known amounts. Relative quantitation also requires comparing your Cq values using a single template molecule.

Conclusion

These are seven tips that can help enhance the efficiency of your PCR. It is crucial to insist on sample quality and product optimization.