Synthesis of cDNA from RNA free of genomic DNA contamination

The synthesis of cDNA is the first step for many protocols in molecular biology, in particular in the analysis of gene expression by PCR and Real-time PCR (qPCR). All subsequent steps require that the synthesis phase produce a highly faithful cDNA that accurately represents the target DNA. The advent of cDNA synthesis kits has made reverse transcription easier and faster with high reproducibility. Two important considerations must be made when choosing a kit for cDNA synthesis: the type of primer and reverse transcriptase. In addition, the RNA must be free from genomic DNA contamination (gDNA). False positives and alterations in the expression of genes are the result of a use of RNA contaminated with gDNA. Hence the need to eliminate gDNA contamination from RNA samples. In vitro digestion with deoxyribonuclease I (DNase I) is the most effective method of removing gDNA contamination in RNA samples. Unfortunately, the removal or inactivation of this enzyme after digestion is problematic.

Generally, methodologies based on high temperatures, the use of chelating agents, the treatment with proteinase K followed by extraction with phenol / chloroform are used to deactivate the enzyme. All these methodologies are not very effective and are often harmful to the integrity of RNA. Some RNA purification kits include a step in which the treatment with DNase is done directly on the spin column. However, this method is not efficient for biological samples that present large quantities of DNA, for which it is necessary to perform the treatment in vitro.

Abmgood’s OneScript cDNA Synthesis kit is a practical and efficient system for single-strand cDNA synthesis. It uses OneScript reverse transcriptase and is equipped with AccuRT, for the elimination of gDNA contaminating RNA samples. AccuRT provided in the OneScript cDNA Synthesis kit effectively removes the contaminating gDNA from the sample in less than 10 minutes, without loss or degradation of RNA. Following this step, the RNA can be retranscribed. The cDNA produced can be used in applications such as PCR and qPCR.

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