Bacterial mRNA Purification by Magnetic Capture‐Hybridization Method

Pang, Xin; Zhou, Dongsheng; Song, Yajun; Pei, Decui; Wang, Jin; Guo, Zhaobiao

doi:10.1111/j.1348-0421.2004.tb03493.x

Cited by 34 publications

(22 citation statements)

References 17 publications

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“…Therefore, prokaryotic rRNAs are often removed before sequencing to improve mRNA detection sensitivity. Different methods have been used to eliminate prokaryotic rRNA, including subtractive hybridization with rRNA-specific probes 7,8 , digestion with exonuclease that preferentially acts on rRNA, poly(A) tail addition to discriminate against rRNA 9,10 , reverse transcription with rRNA-specific primers followed by RNase H digestion to degrade rRNA:DNA hybrids 11 , and gel electrophoresis size separation and extraction of non-rRNA bands 12 .Among these methods, subtractive hybridization and exonuclease digestion have become the most popular owing to the availability of commercial kits from Ambion (MICROBExpress Bacterial mRNA Enrichment kit) and Epicentre (mRNA-ONLY Prokaryotic mRNA Isolation kit). The former kit uses a subtractive hybridization with capture oligonucleotides specific to 16S and 23S rRNAs.…”

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Validation of two ribosomal RNA removal methods for microbial metatranscriptomics

et al. 2010

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The predominance of rRNAs in the transcriptome is a major technical challenge in sequence-based analysis of cDNAs from microbial isolates and communities. Several approaches have been applied to deplete rRNAs from (meta)transcriptomes, but no systematic investigation of potential biases introduced by any of these approaches has been reported. Here we validated the effectiveness and fidelity of the two most commonly used approaches, subtractive hybridization and exonuclease digestion as well as combinations of these treatments, on two synthetic five-microorganism metatranscriptomes using massively parallel sequencing. We found that the effectiveness of rRNA removal was a function of community composition and RNA integrity for these treatments. Subtractive hybridization alone introduced the least bias in relative transcript abundance, whereas exonuclease and in particular combined treatments greatly compromised mRNA abundance fidelity. Illumina sequencing itself also can compromise quantitative data analysis by introducing a G+C bias between runs.Rapid technological advances in ultra-high-throughput sequencing are making de novo sequencing of transcriptomes (RNA-seq) a viable alternative to microarray analysis of microbial isolates and communities 1 . A major technical challenge for de novo transcriptome sequencing is the low relative abundance of mRNAs in total cellular RNA (1-5%; ref.2), the bulk of which is rRNAs and tRNAs 3 . Unlike eukaryotic mRNAs, which can be selectively synthesized into cDNA by virtue of their poly(A) tails 4 , bacterial and archaeal cDNAs are predominantly rRNA -2-sequences 5,6 . Therefore, prokaryotic rRNAs are often removed before sequencing to improve mRNA detection sensitivity. Different methods have been used to eliminate prokaryotic rRNA, including subtractive hybridization with rRNA-specific probes 7,8 , digestion with exonuclease that preferentially acts on rRNA, poly(A) tail addition to discriminate against rRNA 9,10 , reverse transcription with rRNA-specific primers followed by RNase H digestion to degrade rRNA:DNA hybrids 11 , and gel electrophoresis size separation and extraction of non-rRNA bands 12 .Among these methods, subtractive hybridization and exonuclease digestion have become the most popular owing to the availability of commercial kits from Ambion (MICROBExpress Bacterial mRNA Enrichment kit) and Epicentre (mRNA-ONLY Prokaryotic mRNA Isolation kit). The former kit uses a subtractive hybridization with capture oligonucleotides specific to 16S and 23S rRNAs. It has been applied to both bacterial isolates and environmental samples, in one or two rounds 6,[13][14][15][16][17][18] . The Epicentre kit uses exonuclease to preferentially degrade processed RNAs with 5′ monophosphate (the majority of which are believed to be rRNAs) 19,20 . In some instances, these methods have been used in combination to improve rRNA removal [21][22][23] . There is no consensus, however, on the best approach, and existing data are anecdotal. Here we validated the performance of these kits wit...

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Validation of two ribosomal RNA removal methods for microbial metatranscriptomics

et al. 2010

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“…After hybridization, rRNA:probe hybrids bind to beads, allowing their subtraction from the solution. 20 Subtractive hybridization is the most commonly used method so far, partly owning to the availabilities of several commercial kits (Table 3.1).…”

Section: Subtractive Hybridization With Rrna-specific Probesmentioning

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Ribosomal RNA Removal Methods for Microbial Transcriptomics

He¹

2015

Metagenomics for Microbiology

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“…Isolation or enrichment of mRNA is, therefore, an important step in metatranscriptomic experiments. Several methods for mRNA recovery from environmental samples have been described, including: 1) subtractive hybridisation (MICROBExpress Bacterial mRNA Enrichment Kit, Ambion; Pang et al, 2004), 2) exonuclease treatment, which preferentially degrades rRNA (mRNA-ONLY Prokaryotic mRNA Isolation kit, EPICENTRE Biotechnologies, Madison; USA), 3) size separation by gel electrophoresis (McGrath et al, 2008), and 4) duplex specific nuclease (DSN) treatment (Yi et al, 2011). A comparation of two of these approaches (MICROBExpress and DSN) indicated that removal efficiencies vary according to RNA integrity and the environment from which the microbial communities are sampled .…”

Section: Mrna Enrichmentmentioning

confidence: 99%