Description

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.

Features

  • "Genomic DNA degradation step" and "cDNA synthesis step" can be achieved sequentially in approximately 30 min.
  • The master mix reagent contains random and oligo dT primers optimized for efficient reverse transcription.
  • Control, no reverse transcription experiments (no RT-control) can be performed with 5x RT Master Mix II no-RT control.

Applications

cDNA synthesis for real-time PCR

Storage condition

Store at -20ºC

Components

gDNA Remover 10 µl
4x DN Master Mix 440 µl
5x RT Master Mix II 400 µl
5x RT Master Mix II no RT-Control 40 µl
Nuclease-free water 1000 µl × 2

Application data

Example.Efficiency of genomic DNA removal

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".

  4x DN Master Mix 5x RT Master Mix
A gDNA Remover(-) RTase(-)
B gDNA Remover(-) RTase(+)
C gDNA Remover(+) RTase(-)
D gDNA Remover(+) RTase(+)

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".

Template RNA

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

Real-time PCR

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