Comparison of plasma extracellular RNA isolation kits reveals kit-dependent biases.
Author(s): Li X, Mauro M, Williams Z
Publication: Biotechniques, 2015, Vol. 59, Page 13-7
PubMed ID: 26156779 PubMed Review Paper? No
Purpose of Paper
The purpose of this paper was to determine if the RNA extraction kit used affects RNA yield, DNA contamination and RT-PCR amplification of miRNA and mRNA.
Conclusion of Paper
RNA extraction with the RNAdvance kit produced the highest RNA yields, but also the most DNA contamination. Conversely, RNA yield and DNA contamination were the lowest when specimens were extracted with the MagMax kit. RT-PCR amplification success was dependent on the amplicon targeted and the extraction kit used. Generally, RT-PCR amplification of miR-16 and miR-150 yielded the most product when RNA was extracted with miRVana, Direct-zol, miRNeasy, BioFluids or RNAdvance kits and amplification of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and beta actin was most robust when RNA was extracted using the Quick-RNA or miRNeasy kits.
Studies
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Study Purpose
The purpose of this study was to determine if the kit used for RNA extraction affects RNA yield, the level of DNA contamination and RT-PCR amplification efficacy. RNA was extracted from three replicate plasma specimens obtained from a single blood draw. The patient condition was not specified.
Summary of Findings:
While an average of 175.8 ng of RNA was obtained using RNAdvance kit, only 30–40 ng of RNA was obtained using the miRCURY, Biofluids, Quick-RNA, and mirVana kits, and ≤10 ng of RNA was obtained with the DirectZol, miRNeasy and MagMAX kits. DNA contamination was greatest when RNA extraction was performed with the RNAdvance kit, followed by the mirVana and Biofluids kits, although some level of DNA contamination was detected with all 7 kits investigated. The authors report that the lysis buffer used in the RNAdvance kit interfered with the Qubit assay. RT-PCR amplification of miR-16 and miR-150 was the most robust when RNA was extracted with miRVana, Direct-zol, miRNeasy, BioFluids or RNAdvance, while weak amplification was also observed following extraction with Quick-RNA. In contrast, strong RT-PCR amplification of mRNAs beta actin and GAPDH was only possible when RNA was extracted using the Quick-RNA or miRNeasy kit, as weaker amplicons were observed with the RNAdvance, Biofluids or mirVAna kit. Importantly, no amplicons were generated with RNA extracted using the MagMAX kit.
Biospecimens
Preservative Types
- None (Fresh)
Diagnoses:
- Not specified
Platform:
Analyte Technology Platform RNA Real-time qRT-PCR RNA Fluorometry Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Real-time qRT-PCR Specific Targeted nucleic acid miR-16
miR-150
GAPDH
Beta-actin
Analyte Extraction and Purification Analyte isolation method RNAdvance
Biofluids
mirVana
MagMAX
Quick-RNA
DirectZol
miRCURY