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