This kit includes reagents for reverse transcription and for the removal of genomic DNA [DNase I treatment]. In many cases, total RNA prepared using spin-columns or acid guanidium-phenolchloroform (AGPC) extraction methods contains small amount of genomic DNA. Any contaminating genomic DNA will be amplified along with cDNA, especially when primer pairs are designed within the same exon or from pseudogenes. Amplification from genomic DNA can result in qualitative and quantitative inaccuracies. The protocol consists of (i) a genomic DNA degradation step using "gDNA remover" and (ii) a reverse transcription step. The two steps can be achieved sequentially without purification or heat inactivation of DNase I.

cDNA synthesis for real-time PCR

Store at -20ºC

In experiment 1, reverse transcription was performed according to the following condition and Table 1. Then β-actin genes were detected by SYBR® Green I assay. No signal for the "C experiment" indicates that contaminating genomic DNA in the RNA template was completely removed by "gDNA remover".

In experiment 1, reverse transcription was performed according to the following condition and Table 1. Then β-actin genes were detected by SYBR® Green I assay. No signal for the "C experiment" indicates that contaminating genomic DNA in the RNA template was completely removed by "gDNA remover".

Experiment 1: HeLa total RNA 0.5 µg / 10 µL reaction Experiment 2: HeLa total RNA (0, 1 pg, 10 pg, 100 pg, 1 ng, 10 ng, 100 ng, 1 µg) + human gDNA 100 ng / 20 µL reaction

Reagent: THUNDERBIRD™ SYBR® qPCR Mix (Code No. QPS-201) Template: cDNA 2 µL / 20 µL reaction (cDNA solution: 10 %) Target: β-actin (188 bp) Real-time cycler: Applied Biosystems 7900HT