Isolation and amplification of mRNA within a simple microfluidic lab on a chip.
Isolation and amplification of mRNA within a simple microfluidic lab on a chip.
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DOI：
10.1021/ac403417z
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发表时间：
2014-01-07
影响因子：
7.4
通讯作者：
Baeumner, Antje J.
中科院分区：
文献类型：
--
作者：
Reinholt, Sarah J.;Behrent, Arne;Greene, Cassandra;Kalfe, Ayten;Baeumner, Antje J.
开发了微总量分析系统（μTAS）中病原微生物分子生物学检测的主要模块。具体的重点是真核信使RNA （mRNA）的分离和扩增在一个简单的，单通道设备非常低的RNA浓度，然后可以与检测模块集成。小隐孢子虫hsp70 mRNA作为模型分析物。研究的重点是聚甲基丙烯酸甲酯（PMMA）微流控通道内的表面化学，它可以在非常低的mRNA浓度下实现特异性和敏感的mRNA分离和扩增反应。通过UV/臭氧处理将通道表面羧化，然后在其表面固定化聚酰胺胺（PAMAM）树状大分子，从而提高了胸腺嘧啶寡核苷酸oligo(dT)25的固定化效率，并为扩增反应提供了可靠的表面，重要的是，不需要阻断剂。研究人员对剩余活性表面基团进行了额外的化学修饰，以避免非特异性捕获核酸和在低RNA浓度下阻碍mRNA扩增。mRNA的扩增使用基于核酸序列的扩增（NASBA）完成，这是一种等温，引物依赖的技术。由先前产生的NASBA扩增子组成的阳性对照可以稀释1015倍，仍然可以成功地在片上重新扩增。最后，直接在芯片上演示了从30个小卵囊中成功分离和扩增mRNA的方法，并与台式设备进行了比较。这是在一个简单的微流控装置内成功分离mRNA和基于nasba的mRNA扩增的第一个证据。
The major modules for realizing molecular biological assays in a micro total analysis system (μTAS) were developed for the detection of pathogenic organisms. The specific focus was the isolation and amplification of eukaryotic messenger RNA (mRNA) within a simple, single-channel device for very low RNA concentrations that could then be integrated with detection modules. The hsp70 mRNA from Cryptosporidium parvum was used as a model analyte. Important points of study were surface chemistries within poly(methyl methacrylate) (PMMA) microfluidic channels that enabled specific and sensitive mRNA isolation and amplification reactions for very low mRNA concentrations. Optimal conditions were achieved when the channel surface was carboxylated via UV/ozone treatment followed by the immobilization of polyamidoamine (PAMAM) dendrimers on the surface, thus increasing the immobilization efficiency of the thymidine oligonucleotide, oligo(dT)25, and providing a reliable surface for the amplification reaction, importantly, without the need for blocking agents. Additional chemical modifications of the remaining active surface groups were studied to avoid non-specific capturing of nucleic acids and hindering of the mRNA amplification at low RNA concentrations. Amplification of the mRNA was accomplished using nucleic acid sequence-based amplification (NASBA), an isothermal, primer-dependent technique. Positive controls consisting of previously generated NASBA amplicons could be diluted 1015 fold and still result in successful on-chip re-amplification. Finally, the successful isolation and amplification of mRNA from as few as 30 C. parvum oocysts was demonstrated directly on-chip and compared to bench-top devices. This is the first proof of successful mRNA isolation and NASBA-based amplification of mRNA within a simple microfluidic device in relevant analytical volumes.
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影响因子：
2.8
作者：
Mahalanabis M;Do J;ALMuayad H;Zhang JY;Klapperich CM
通讯作者：
Klapperich CM
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影响因子：
13.3
作者：
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通讯作者：
Yarmush, Martin L.
影响因子：
7.4
作者：
Bhattacharyya, A;Klapperich, CM
通讯作者：
Klapperich, CM
影响因子：
6.1
作者：
Goral, VN;Zaytseva, NV;Baeumner, AJ
通讯作者：
Baeumner, AJ
影响因子：
7.4
作者：
Hagan, Kristin A.;Bienvenue, Joan M.;Landers, James P.
通讯作者：
Landers, James P.