Overview

Norgen’s Total RNA Purification Maxi Kit provides a rapid method for the isolation and purification of total RNA from cultured animal cells, tissue samples, blood, bacteria, yeast, fungi, plants, and viruses. The kit purifies all sizes of RNA, from large mRNA and ribosomal RNA down to microRNA (miRNA) and small interfering RNA (siRNA). The RNA is preferentially purified from other cellular components such as proteins, without the use of phenol or chloroform. The purified RNA is of the highest integrity, and can be used in a number of downstream applications including real-time PCR, reverse transcription PCR, Northern blotting, RNase protection and primer extension, and expression array assays.

Norgen’s Purification Technology

Purification is based on spin column chromatography using Norgen’s proprietary resin as the separation matrix. The RNA is preferentially purified from other cellular components such as proteins without the use of phenol or chloroform. The process involves first lysing the cells or tissue of interest with the provided Buffer RL (please see the flow chart on page 4). Ethanol is then added to the lysate, and the solution is loaded onto a maxi spin column. Norgen’s resin binds RNA in a manner that depends on ionic concentrations. Thus only the RNA will bind to the column, while the contaminating proteins will be removed in the flowthrough or retained on the top of the resin. The bound RNA is then washed with the provided Wash Solution A in order to remove any remaining impurities, and the purified total RNA is eluted with the Elution Solution A. The purified RNA is of the highest integrity, and can be used in a number of downstream applications.

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Supporting Data

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Figure 1. Isolate a Diversity of RNA Species. Total RNA was isolated from 10 mL of an overnight E. coli culture using Norgen's Total RNA Purification Maxi Kit. One microliter of the 2 mL elution was analyzed on an Agilent® 2100 BioAnalyzer RNA Nano 6000 chip. As it can be seen, high-quality total RNA was isolated with minimal degradation. As well, a large amount of small RNA (< 200 nt) was recovered, indicating that the kit is able to isolate a diversity of RNA species.

Figure 2. Good Amplification and Consistency of Isolation. Norgen's Total RNA Purification Maxi Kit was used to isolate total RNA from six different overnight samples of E. coli (10 mL samples). In brief, one microgram of the isolated RNA was subjected to a 20 µL reverse transcription using Invitrogen Superscript III. Three microliters of the reverse transcription were used as the template in a 20 µL qPCR reaction using 16S rRNA E. coli specific primers and Bio-Rad iQ mix. As it can be seen, the RNA was detected and successfully amplified in all cases. Furthermore, all samples showed a similar Ct value, indicating consistent isolation.

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