Guidelines for Successful Quantitative Gene Expression in Real- Time qPCR Assays
The qPCR is an efficient power tool to ensure the mRNA expression in several kinds of samples. To obtain reliable results, numerous parameters should be considered, including: (a) a good quality RNA; (b) specific and efficient primers; (c) appropriate dyes or probes according to the analysis; (d) stable reference genes with respect to analyzed condition; (e) normalization of expression; and (f) a combined approach of available software. Finally, we highlight that adopting all described guidelines, the possible errors and wrong procedures will be decreased, thus rendering successfully the results in real-time qPCR assays.
Quality of RNA
High quality of RNA is an essential requirement for qPCR. There are several probable contaminants that may interfere in PCR reactions by inhibiting mainly transcriptase reverse and DNA polymerases enzymes, such as DNA genomic, excess of proteins and carbohydrates, as well as phenolic compounds.
An additional purification step must be performed before starting qPCR reactions, digesting the genomic DNA; on the contrary, the DNA can act as template during qPCR and produce unreliable results. It may be avoided employing RNase-free DNase enzymes directly in the samples of RNA or without treatments but using specific primers designed in the exon-exon boundary of gene coding region.
Primer design and probe considerations
Designing specific primers and adopting appropriate probes are crucial requirements for amplification efficiency, specificity and fluorescence in qPCR assays. The primer should be designed in junction exon-exon of genic sequence to avoid amplification of contaminant genomic DNA, amplifying specifically the target cDNA sequence
Importance of the reference genes to normalize qPCR data
Reference or constitutive genes are required to normalize the target gene-expression quantification in qPCR assays. The normalization of expression levels is pivotal once it avoids misinterpretation of data obtained in qPCR reactions. Thus, a group of constitutive genes should be analyzed by stability, choosing the most stable ones as reference to be used in the data normalization.
Accuracy of relative gene expression can be severely affected by a wrong choice of reference genes to normalize and validate the final results; consequently, employing inappropriate genes as reference for data normalization may lead to erroneous results and data misinterpretation. Thus, the expression stability of a reference gene must be confirmed in each experimental condition before the qPCR assays and it should be taking into account that a unique gene is generally not suitable for normalization.
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