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Comparative Study
. 2011:4:3.
doi: 10.1186/1756-0500-4-3. Epub 2011 Jan 6.

Comparison of different methods for DNA-free RNA isolation from SK-N-MC neuroblastoma

Comparative Study

Comparison of different methods for DNA-free RNA isolation from SK-N-MC neuroblastoma

Lucélia Tavares et al. BMC Res Notes. 2011.

Abstract

Background: RNA quality and quantity are important factors for ensuring the accuracy of gene expression analysis and other RNA-based downstream applications. Extraction of high quality nucleic acids is difficult from neuronal cells and brain tissues as they are particularly rich in lipids. In addition, most common RNA extraction methods are phenol-based, resulting in RNA that may be incompatible with downstream applications such as gene expression.

Findings: In this work, a comparative analysis of the RNA quality obtained from SK-N-MC cells was performed using six commonly used RNA isolation kits: two phenol-based kits and four non-phenol based kits. The non-phenol based kits tested AxyPrep Multisource Total RNA Miniprep, RNeasy Mini, EasySpin and Ilustra RNAspin Mini RNA Isolation, all performed well and resulted in the isolation of high quality RNA, as evaluated by A260/A280. The RNA extracted with AxyPrep Multisource Total RNA Miniprep, RNeasy Mini and EasySpin provided the highest RNA yields. In particular, the RNA isolated by AxyPrep Multisource Total RNA Miniprep Kit did not show any detectable genomic DNA contamination even without previous DNase treatment or after RNA direct PCR amplification using universal 18S primers.

Conclusions: The RNA extracted from SK-N-MC cells with AxyPrep Multisource Total RNA Miniprep Kit was superior with respect to the RNA quality and concentration. This kit does not use aggressive organic solvents and RNA free of genomic DNA was isolated without the need for DNase treatment.

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Figures

Figure 1
Figure 1
Electrophoresis of RNA samples in 2% (w/v) agarose gel, stained with ethidium bromide. L- 100 bp ladder; 1- RNA (untreated sample) isolated by AxyPrep Multisource Total RNA Miniprep kit (Axygen); 2- RNA (treated with Turbo™ DNase, Ambion) isolated by AxyPrep Multisource Total RNA Miniprep kit (Axygen); 3- RNA (DNase I untreated sample) isolated by RNeasy® Mini kit (Qiagen); 4- RNA (treated with Turbo™ DNase, Ambion) isolated by RNeasy® Mini kit (Qiagen).
Figure 2
Figure 2
Electrophoresis of 18S PCR amplification products of RNA samples without reverse transcription, in 2% (w/v) agarose gel, stained with ethidium bromide. 18S PCR amplification products from RNA isolated by AxyPrep Multisource Total RNA Miniprep Kit (Axygen); 2-18S PCR amplification from RNA isolated by AxyPrep Multisource Total RNA Miniprep Kit (Axygen) and treated with Turbo™ DNase (Ambion); 3-18S PCR amplification products from RNA isolated by RNeasy® Mini Kit (Qiagen); 4-18S PCR amplification products from RNA isolated by RNeasy® Mini Kit (Qiagen) and treated with Turbo™ DNase (Ambion); 5-18S PCR amplification products from water (negative control); L- 100 bp ladder.

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