TY - JOUR
T1 - Optimization of whole-transcriptome amplification from low cell density deep-sea microbial samples for metatranscriptomic analysis
AU - Wu, Jieying
AU - Gao, Weimin
AU - Zhang, Weiwen
AU - Meldrum, Deirdre
N1 - Funding Information:
We would like to thank the crew of the R/V Thompson TN221 Research Cruise with chief scientists Dr. John Delaney and Dr. Deborah Kelley of the University of Washington, and Dr. Joseph Shih-Hui Chao and Dr. Cody Youngbull from the Center for Biosignatures Discovery Automation in the Biodesign Institute at Arizona State University (ASU) for their help with the water sampling. We also thank ASU for the support of this research.
PY - 2011/1
Y1 - 2011/1
N2 - Limitation in sample quality and quantity is one of the big obstacles for applying metatranscriptomic technologies to explore gene expression and functionality of microbial communities in natural environments. In this study, several amplification methods were evaluated for whole-transcriptome amplification of deep-sea microbial samples, which are of low cell density and high impurity. The best amplification method was identified and incorporated into a complete protocol to isolate and amplify deep-sea microbial samples. In the protocol, total RNA was first isolated by a modified method combining Trizol (Invitrogen, CA) and RNeasy (QIAGEN, CA) method, amplified with a WT-Ovation™ Pico RNA Amplification System (NuGEN, CA), and then converted to double-strand DNA from single-strand cDNA with a WT-Ovation™ Exon Module (NuGEN, CA). The products from the whole-transcriptome amplification of deep-sea microbial samples were assessed first through random clone library sequencing. The BLAST search results showed that marine-based sequences are dominant in the libraries, consistent with the ecological source of the samples. The products were then used for next-generation Roche GS FLX Titanium sequencing to obtain metatranscriptome data. Preliminary analysis of the metatranscriptomic data showed good sequencing quality. Although the protocol was designed and demonstrated to be effective for deep-sea microbial samples, it should be applicable to similar samples from other extreme environments in exploring community structure and functionality of microbial communities.
AB - Limitation in sample quality and quantity is one of the big obstacles for applying metatranscriptomic technologies to explore gene expression and functionality of microbial communities in natural environments. In this study, several amplification methods were evaluated for whole-transcriptome amplification of deep-sea microbial samples, which are of low cell density and high impurity. The best amplification method was identified and incorporated into a complete protocol to isolate and amplify deep-sea microbial samples. In the protocol, total RNA was first isolated by a modified method combining Trizol (Invitrogen, CA) and RNeasy (QIAGEN, CA) method, amplified with a WT-Ovation™ Pico RNA Amplification System (NuGEN, CA), and then converted to double-strand DNA from single-strand cDNA with a WT-Ovation™ Exon Module (NuGEN, CA). The products from the whole-transcriptome amplification of deep-sea microbial samples were assessed first through random clone library sequencing. The BLAST search results showed that marine-based sequences are dominant in the libraries, consistent with the ecological source of the samples. The products were then used for next-generation Roche GS FLX Titanium sequencing to obtain metatranscriptome data. Preliminary analysis of the metatranscriptomic data showed good sequencing quality. Although the protocol was designed and demonstrated to be effective for deep-sea microbial samples, it should be applicable to similar samples from other extreme environments in exploring community structure and functionality of microbial communities.
KW - Amplification
KW - Deep-sea microbial samples
KW - Whole-transcriptome
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U2 - 10.1016/j.mimet.2010.10.018
DO - 10.1016/j.mimet.2010.10.018
M3 - Article
C2 - 21044647
AN - SCOPUS:78650195187
SN - 0167-7012
VL - 84
SP - 88
EP - 93
JO - Journal of Microbiological Methods
JF - Journal of Microbiological Methods
IS - 1
ER -