A Lab-on-a-chip for Rapid MiRNA Extraction

Overview

Journal PLoS One

Specialties General Medicine
Science

Date 2019 Dec 20

PMID 31856234

Citations 4

Authors

Ole Behrmann

Matthias Hugle

Peter Bronsert

Bettina Herde

Julian Heni

Marina Schramm

Frank T Hufert

Gerald A Urban

Gregory Dame

Affiliations

Soon will be listed here.

Abstract

We present a simple to operate microfluidic chip system that allows for the extraction of miRNAs from cells with minimal hands-on time. The chip integrates thermoelectric lysis (TEL) of cells with native gel-electrophoretic elution (GEE) of released nucleic acids and uses non-toxic reagents while requiring a sample volume of only 5 μl. These properties as well as the fast process duration of 180 seconds make the system an ideal candidate to be part of fully integrated point-of-care applications for e.g. the diagnosis of cancerous tissue. GEE was characterized in comparison to state-of-the-art silica column (SC) based RNA recovery using the mirVana kit (Ambion) as a reference. A synthetic miRNA (miR16) as well as a synthetic snoRNA (SNORD48) were subjected to both GEE and SC. Subsequent detection by stem-loop RT-qPCR demonstrated a higher yield for miRNA recovery by GEE. SnoRNA recovery performance was found to be equal for GEE and SC, indicating yield dependence on RNA length. Coupled operation of the chip (TEL + GEE) was characterized using serial dilutions of 5 to 500 MCF7 cancer cells in suspension. Samples were split and cells were subjected to either on-chip extraction or SC. Eluted miRNAs were then detected by stem-loop RT-qPCR without any further pre-processing. The extraction yield from cells was found to be up to ~200-fold higher for the chip system under non-denaturing conditions. The ratio of eluted miRNAs is shown to be dependent on the degree of complexation with miRNA associated proteins by comparing miRNAs purified by GEE from heat-shock and proteinase-K based lysis.

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References

Aravin A, Tuschl T . Identification and characterization of small RNAs involved in RNA silencing. FEBS Lett. 2005; 579(26):5830-40. DOI: 10.1016/j.febslet.2005.08.009. View

Catto J, Alcaraz A, Bjartell A, de Vere White R, Evans C, Fussel S . MicroRNA in prostate, bladder, and kidney cancer: a systematic review. Eur Urol. 2011; 59(5):671-81. DOI: 10.1016/j.eururo.2011.01.044. View

Redshaw N, Wilkes T, Whale A, Cowen S, Huggett J, Foy C . A comparison of miRNA isolation and RT-qPCR technologies and their effects on quantification accuracy and repeatability. Biotechniques. 2013; 54(3):155-64. DOI: 10.2144/000114002. View

Hugle M, Dame G, Behrmann O, Rietzel R, Karthe D, Hufert F . A lab-on-a-chip for preconcentration of bacteria and nucleic acid extraction. RSC Adv. 2022; 8(36):20124-20130. PMC: 9080779. DOI: 10.1039/c8ra02177e. View

Gregory R, Chendrimada T, Cooch N, Shiekhattar R . Human RISC couples microRNA biogenesis and posttranscriptional gene silencing. Cell. 2005; 123(4):631-40. DOI: 10.1016/j.cell.2005.10.022. View

Heneghan H, Miller N, Kelly R, Newell J, Kerin M . Systemic miRNA-195 differentiates breast cancer from other malignancies and is a potential biomarker for detecting noninvasive and early stage disease. Oncologist. 2010; 15(7):673-82. PMC: 3228012. DOI: 10.1634/theoncologist.2010-0103. View

Shintaku H, Nishikii H, Marshall L, Kotera H, Santiago J . On-chip separation and analysis of RNA and DNA from single cells. Anal Chem. 2014; 86(4):1953-7. DOI: 10.1021/ac4040218. View

Schoch R, Ronaghi M, Santiago J . Rapid and selective extraction, isolation, preconcentration, and quantitation of small RNAs from cell lysate using on-chip isotachophoresis. Lab Chip. 2009; 9(15):2145-52. DOI: 10.1039/b903542g. View

Arzt L, Kothmaier H, Quehenberger F, Halbwedl I, Wagner K, Maierhofer T . Evaluation of formalin-free tissue fixation for RNA and microRNA studies. Exp Mol Pathol. 2011; 91(2):490-5. DOI: 10.1016/j.yexmp.2011.05.007. View

10.

Calin G, Croce C . MicroRNA signatures in human cancers. Nat Rev Cancer. 2006; 6(11):857-66. DOI: 10.1038/nrc1997. View

11.

Bravo V, Rosero S, Ricordi C, Pastori R . Instability of miRNA and cDNAs derivatives in RNA preparations. Biochem Biophys Res Commun. 2007; 353(4):1052-5. DOI: 10.1016/j.bbrc.2006.12.135. View

12.

Meister G, Landthaler M, Patkaniowska A, Dorsett Y, Teng G, Tuschl T . Human Argonaute2 mediates RNA cleavage targeted by miRNAs and siRNAs. Mol Cell. 2004; 15(2):185-97. DOI: 10.1016/j.molcel.2004.07.007. View

13.

Burgos K, Javaherian A, Bomprezzi R, Ghaffari L, Rhodes S, Courtright A . Identification of extracellular miRNA in human cerebrospinal fluid by next-generation sequencing. RNA. 2013; 19(5):712-22. PMC: 3677285. DOI: 10.1261/rna.036863.112. View

14.

Motameny S, Wolters S, Nurnberg P, Schumacher B . Next Generation Sequencing of miRNAs - Strategies, Resources and Methods. Genes (Basel). 2014; 1(1):70-84. PMC: 3960865. DOI: 10.3390/genes1010070. View

15.

Gee H, Buffa F, Camps C, Ramachandran A, Leek R, Taylor M . The small-nucleolar RNAs commonly used for microRNA normalisation correlate with tumour pathology and prognosis. Br J Cancer. 2011; 104(7):1168-77. PMC: 3068486. DOI: 10.1038/sj.bjc.6606076. View

16.

Zhou D, Du W, Xi Q, Ge J, Jiang J . Isothermal nucleic acid amplification strategy by cyclic enzymatic repairing for highly sensitive microRNA detection. Anal Chem. 2014; 86(14):6763-7. DOI: 10.1021/ac501857m. View

17.

Fromm B, Billipp T, Peck L, Johansen M, Tarver J, King B . A Uniform System for the Annotation of Vertebrate microRNA Genes and the Evolution of the Human microRNAome. Annu Rev Genet. 2015; 49:213-42. PMC: 4743252. DOI: 10.1146/annurev-genet-120213-092023. View

18.

Kramer M . Stem-loop RT-qPCR for miRNAs. Curr Protoc Mol Biol. 2011; Chapter 15:Unit 15.10. PMC: 3152947. DOI: 10.1002/0471142727.mb1510s95. View

19.

Chen C, Ridzon D, Broomer A, Zhou Z, Lee D, Nguyen J . Real-time quantification of microRNAs by stem-loop RT-PCR. Nucleic Acids Res. 2005; 33(20):e179. PMC: 1292995. DOI: 10.1093/nar/gni178. View

20.

Rajput S, Dave V, Rajput A, Pandey H, Datta T, Singh R . A column-based rapid method for the simultaneous isolation of DNA, RNA, miRNA and proteins. Cell Biol Int. 2012; 36(9):779-83. DOI: 10.1042/CBI20110342. View