Avraham R, Haseley N, Fan A, Bloom-Ackermann Z, Livny J, Hung DT (2016) A highly multiplexed and sensitive RNA-seq protocol for simultaneous analysis of host and pathogen transcriptomes. Nature Protocols 11: 8, 1477-1491. doi:10.1038/nprot.2016.090

The ability to simultaneously characterize the bacterial and host expression programs during infection would facilitate a comprehensive understanding of pathogen–host interactions. Although RNA sequencing (RNA-seq) has greatly advanced our ability to study the transcriptomes of prokaryotes and eukaryotes separately, limitations in existing protocols for the generation and analysis of RNA-seq data have hindered simultaneous profiling of host and bacterial pathogen transcripts from the same sample.

This study provides a detailed protocol for simultaneous analysis of host and bacterial transcripts by RNA-seq. Importantly, this protocol details the steps required for efficient host and bacteria lysis, barcoding of samples, technical advances in sample preparation for low-yield sample inputs and a computational pipeline for analysis of both mammalian and microbial reads from mixed host–pathogen RNA-seq data. Sample preparation takes 3 d from cultured cells to pooled libraries. Data analysis takes an additional day. Compared with previous methods, the protocol detailed here provides a sensitive, facile and generalizable approach that is suitable for large-scale studies and will enable the field to obtain in-depth analysis of host–pathogen interactions in infection models.

An extraction mix was made from 2 µl of silver buffer (from the RNAGEM kit), 1 µl of RNAGEM and a cell pellet of up to 17 µl, giving a total extraction mix volume of 20 µl.

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RNAGEM

Temperature-driven, single-tube extractions of total RNA from mammalian cells, tissues, insects, bacteria and virus

This powerful, broad-specificity approach is ideal for gene expression from a very small number of cells, such as exosomes or single cells, without the need for harsh chemicals or further purification.

  • Ideal for preparing RNA from mammalian cell culture, laser capture microdissection, FACS-prepared cell populations, cells in suspension, adherent cells, cells stored in RNAlaterTM and cell pellets
  • Simplified, hands-off workflow provides RT-PCR and RT-qPCR ready RNA in approximately 5 minutes
  • Releases both RNA and DNA with excellent recovery across a wide range of cell numbers
  • Requires no further purification for accurate RT-PCR and RT-qPCR analysis
  • Inhibitor-free means no harsh chemical washes or multiple steps
  • RT-PCR and RT-qPCR reagents can be added directly to the lysate for a simple, streamlined protocol in 96 well plates
  • Each kit contains RNAGEM, DNase I, BLUE buffer, 10x DNase buffer, and 10x TE

Learn more about RNAGEM and recent applications. Download our RNAGEM technical overview. Watch our webinar on advances in extracting and stabilizing RNA.

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