A comparison of commercially-available automated and manual extraction kits for the isolation of total RNA from small tissue samples

BMC Biotechnology

Marlo K Sellin Jeffries²  Andor J Kiss¹  Austin W Smith²  James T Oris² 

[1] Center for Bioinformatics and Functional Genomics, Miami University, Oxford 45056, OH, USA

[2] Department of Biology, Miami University, Oxford 45056, OH, USA

关键词: Fathead minnow;    Small tissues;    Microarray;    Next generation sequencing;    RNA-Seq;    qPCR;    Gene expression;    RNA extraction;   

Others  :  1084450
DOI  :  10.1186/s12896-014-0094-8

 received in 2014-05-20, accepted in 2014-10-22,  发布年份 2014

【 摘 要 】

Background

This study compared the performance of five commercially available kits in extracting total RNA from small eukaryotic tissue samples (<15 mg). Total RNA was isolated from fathead minnow (Pimephales promelas) tissues (spleen, blood, kidney, embryo, and larvae) using the Qiagen RNeasy® Plus Mini, Qiagen RNeasy® Plus Universal, Promega Maxwell® 16 LEV simplyRNA, Ambion MagMAX™-96 and Promega SimplyRNA HT kits. Kit performance was evaluated via measures of RNA quantity (e.g., total RNA amount) and quality (e.g., ratio of absorbance at 260 and 280 nm, RNA integrity number (RIN), presence of gDNA).

Results

With the exception of embryos, each kit generally extracted ≥5 μg of total RNA from each sample. With regard to RNA quality, the RINs of RNA samples isolated via the Plus Mini and Maxwell® 16 kits were consistently higher than those of samples extracted via the remaining three kits and for all tissues, these kits produced intact RNA with average RIN values ≥7. The Plus Universal and SimplyRNA HT kits produced moderately degraded (RIN values <7, but ≥5), while the RNA recovered via the MagMAX™ kit tended to exhibit a high degree of degradation (RIN values <5).

Conclusions

Each kit was generally capable of extracting the amount of RNA required for most downstream gene expression applications suggesting that RNA yield is unlikely to be a limiting factor for any of the kits evaluated. However, differences in the quality of RNA extracted via each of the kits indicate that these kits may differ in their ability to yield RNA acceptable for some applications. Overall, the findings of this study demonstrate that there are practical differences between commercially available RNA extraction kits that should be taken into account when selecting extraction methods to be used for isolating RNA designated for gene expression analysis.

【 授权许可】

   
2014 Sellin Jeffries et al.; licensee BioMed Central Ltd.

【 图 表 】

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