How can amplification of genomic DNA in total RNA samples be avoided?
How can amplification of genomic DNA in total RNA samples be avoided?
To avoid amplification of genomic DNA in total RNA samples:
*Design primers that avoid genomic amplification: select a large intron region and design forward and reverse primers in exons upstream and downstream of the intron. With this strategy, genomic amplifications cannot occur for large introns. When introns are small, genomic amplification can be differentiated based on differences in melting temperature as the result of different amplification sizes (melt curve).
*For genes that lack introns or when the genomic structure is unknown, treat the total RNA with DNase I to remove genomic DNA.
*Use the PrimeScript RT Reagent Kit with gDNA Eraser (Perfect Real Time) (Cat.# RR047A). The gDNA Eraser in this kit removes genomic DNA in just 2 minutes, followed by a 15-minute reverse transcription reaction. The resulting cDNA can be amplified using a Takara Bio qPCR premix.