Overview

This kit provides for rapid spin column isolation and purification of total RNA and genomic DNA simultaneously from a single plant sample without splitting the lysate. Norgen's plant lysis solution is highly robust and effective over a wide range of plants including challenging samples.  The total RNA and genomic DNA are both column purified in under 30 minutes.   Since RNA and DNA are isolated without splitting the lysate, variability and inconsistent results are reduced.​​ All sizes of RNA including microRNA are recovered without the need for phenol.  Optional on-column DNase and RNase treatments provide flexibility to isolate DNA-free RNA or RNA-free DNA respectively.  Isolated nucleic acids are of a high quality and yield, and are ready for downstream use including PCR, qPCR, RT-PCR, qRT-PCR, sequencing and more. 

Background

It is often necessary to isolate total RNA and genomic DNA from a single plant sample, such as for studies of gene expression, mutant or transgenic plant characterization, and host plant-pathogen characterization. This is of great benefit when isolating RNA and DNA from precious, difficult to obtain or very small samples. Furthermore, gene expression analysis will be more reliable since the RNA and DNA are derived from the same sample, therefore eliminating inconsistent results.

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Supporting Data

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Figure 1. Isolation of Total RNA and Genomic DNA from Tobacco, Tomato and Peach Leaf Tissue. Total RNA and genomic DNA were isolated from 50 mg of tobacco leaf, 50 mg of tomato leaf and 50 mg of peach leaf using Norgen's Plant RNA/DNA Purification Kit. Panel A is a 1X MOPS 1.5% agarose gel showing the total RNA that was isolated after the optional on-column DNase digestion. 5 µL of total RNA from each 75 µL elution was mixed with 2x RNA loading dye and denatured at 70°C for 10 minutes and loaded onto the gel. Lane M is Norgen's 1 kb RNA Ladder, Lanes 1 and 2 contain RNA isolated from tobacco cells, Lanes 3 and 4 contain RNA isolated from tomato cells, and Lanes 5 and 6 contain RNA isolated from peach cells. Panel B is a 1.5% agarose gel containing the genomic DNA that was isolated after the optional on-column RNase digestion, and in each case 10 µL of the 75 µL elution was loaded. Lane M is Norgen's HighRanger 1kb DNA Ladder, Lanes 1 and 2 contain the tobacco DNA, Lanes 3 and 4 contain the tomato DNA, and Lanes 5 and 6 contain the peach DNA. The RNA and DNA are intact and of the highest quality, and can be used in a number of different downstream applications.

Figure 2. Detection of 18s rDNA by Real-time PCR (SYBR Green). Total DNA and RNA from 50 mg of tobacco, tomato and peach were simultaneously isolated with Norgen's Plant RNA/DNA Purification Kit (with the optional on-column RNase treatment). Next, 2 µL of DNA from each 75 µL elution was mixed in 20 µL of PCR reaction and used in a real-time PCR (95°C for 3 minutes and 40 cycles at 95°C for 15 seconds and 60°C for 30 seconds.). DNA from tobacco (green), tomato (red) and peach (blue) was successfully amplified, indicating the high quality of the DNA for downstream application. Primer dimer (black) also was shown.

Figure 3. Isolate High Quality Total RNA from Challenging Samples. Total RNA was purified from 50 mg of grape and peach leaves using Norgen's Plant RNA/DNA Purification Kit, with the optional on-column DNase treatment. Next, 1 µL of 75 µL eluted RNA was resolved on an Agilent 2100 bioanalyzer using an RNA Nano 6000 chip. High quality total RNA was isolated from both samples, even from the challenging grape leaf sample.

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