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

Tavares L, Alves PM, Ferreira RB, Santos CN - BMC Res Notes (2011)

Bottom Line: 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.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.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.

View Article: PubMed Central - HTML - PubMed

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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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).
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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).

Mentions: DNase I digestion has consistently proven to be the most effective method for removing DNA contamination from RNA samples. DNase I treatment efficacy test was evaluated for the two best performing kits; RNA extracted with AxyPrep Multisource, Total RNA Miniprep and RNeasy® Mini. Results from kits, with or without DNase treatment, were visualized in agarose gel stained with ethidium bromide (Figure 1). RNA isolated with AxyPrep Multisource Total RNA Miniprep kit did not show visible genomic DNA contamination even without DNase treatment (Figure 1, lanes 1 and 2). On the contrary, RNA extracted using the RNeasy® Mini Kit clearly contained DNA contamination, which disappeared promptly after DNase treatment. Very low levels of DNA contamination, albeit not detectable by agarose gel electrophoresis, may be amplified and then corrupt the results obtained by highly sensitive techniques such as Real-Time PCR. In an attempt to address and evaluate this hypothesis, RNA samples (with and without DNase treatment) isolated by Axygen and Qiagen kits were directly amplified by PCR using universal 18S primers. The corresponding reaction products where visualized by agarose gel electrophoresis (Figure 2). Although not visualized by direct RNA electrophoresis in Figure 1, RNA isolated using the RNeasy® Mini kit and treated with DNase was shown to contain DNA contamination after PCR amplification (Figure 2, lanes 3 and 4). This result was obtained after confirming experimentally that the PCR product amplification was a result of the RNA concentration in the sample (and therefore the DNA contaminant amount) and was not due to extensive amplification (results not shown). After direct PCR amplification, the RNA isolated by AxyPrep Multisource Total RNA Miniprep kit (with or without previous DNase treatment) did not reveal the presence of any band on the agarose gel (Figure 2, lanes 1 and 2). Therefore, the Axygen kit provides reliable and good quality RNA isolation from SK-N-MC neuroblastoma cells, thus suitable for a successful RNA amplification without the need of any DNase treatment. DNase treatment is considered disadvantageous by some investigators, as it adds extra salts and protein to the sample and can affect the efficiency of the subsequent cDNA synthesis.


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

Tavares L, Alves PM, Ferreira RB, Santos CN - BMC Res Notes (2011)

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).
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3050700&req=5

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).
Mentions: DNase I digestion has consistently proven to be the most effective method for removing DNA contamination from RNA samples. DNase I treatment efficacy test was evaluated for the two best performing kits; RNA extracted with AxyPrep Multisource, Total RNA Miniprep and RNeasy® Mini. Results from kits, with or without DNase treatment, were visualized in agarose gel stained with ethidium bromide (Figure 1). RNA isolated with AxyPrep Multisource Total RNA Miniprep kit did not show visible genomic DNA contamination even without DNase treatment (Figure 1, lanes 1 and 2). On the contrary, RNA extracted using the RNeasy® Mini Kit clearly contained DNA contamination, which disappeared promptly after DNase treatment. Very low levels of DNA contamination, albeit not detectable by agarose gel electrophoresis, may be amplified and then corrupt the results obtained by highly sensitive techniques such as Real-Time PCR. In an attempt to address and evaluate this hypothesis, RNA samples (with and without DNase treatment) isolated by Axygen and Qiagen kits were directly amplified by PCR using universal 18S primers. The corresponding reaction products where visualized by agarose gel electrophoresis (Figure 2). Although not visualized by direct RNA electrophoresis in Figure 1, RNA isolated using the RNeasy® Mini kit and treated with DNase was shown to contain DNA contamination after PCR amplification (Figure 2, lanes 3 and 4). This result was obtained after confirming experimentally that the PCR product amplification was a result of the RNA concentration in the sample (and therefore the DNA contaminant amount) and was not due to extensive amplification (results not shown). After direct PCR amplification, the RNA isolated by AxyPrep Multisource Total RNA Miniprep kit (with or without previous DNase treatment) did not reveal the presence of any band on the agarose gel (Figure 2, lanes 1 and 2). Therefore, the Axygen kit provides reliable and good quality RNA isolation from SK-N-MC neuroblastoma cells, thus suitable for a successful RNA amplification without the need of any DNase treatment. DNase treatment is considered disadvantageous by some investigators, as it adds extra salts and protein to the sample and can affect the efficiency of the subsequent cDNA synthesis.

Bottom Line: 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.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.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.

View Article: PubMed Central - HTML - PubMed

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.

Show MeSH
Related in: MedlinePlus