Global Array-Based Transcriptomics from Minimal Input RNA Utilising an Optimal RNA Isolation Process Combined with SPIA cDNA Probes

Laura Kennedy, Maheshwar Pauriah, Valerie Godfrey, Jacqueline Howie, Helen Dennis, Daniel Crowther, Allan Struthers, Catharine Goddard, Giora Feuerstein, Chim Lang, Gino Miele

    Research output: Contribution to journalArticle

    3 Citations (Scopus)
    93 Downloads (Pure)

    Abstract

    Background: Technical advances in the collection of clinical material, such as laser capture microdissection and cell sorting, provide the advantage of yielding more refined and homogenous populations of cells. However, these attractive advantages are counter balanced by the significant difficultly in obtaining adequate nucleic acid yields to allow transcriptomic analyses. Established technologies are available to carry out global transcriptomics using nanograms of input RNA, however, many clinical samples of low cell content would be expected to yield RNA within the picogram range. To fully exploit these clinical samples the challenge of isolating adequate RNA yield directly and generating sufficient microarray probes for global transcriptional profiling from this low level RNA input has been addressed in the current report. We have established an optimised RNA isolation workflow specifically designed to yield maximal RNA from minimal cell numbers. This procedure obtained RNA yield sufficient for carrying out global transcriptional profiling from vascular endothelial cell biopsies, clinical material not previously amenable to global transcriptomic approaches. In addition, by assessing the performance of two linear isothermal probe generation methods at decreasing input levels of good quality RNA we demonstrated robust detection of a class of low abundance transcripts (GPCRs) at input levels within the picogram range, a lower level of RNA input (50 pg) than previously reported for global transcriptional profiling and report the ability to interrogate the transcriptome from only 10 pg of input RNA. By exploiting an optimal RNA isolation workflow specifically for samples of low cell content, and linear isothermal RNA amplification methods for low level RNA input we were able to perform global transcriptomics on valuable and potentially informative clinically derived vascular endothelial biopsies here for the first time. These workflows provide the ability to robustly exploit ever more common clinical samples yielding extremely low cell numbers and RNA yields for global transcriptomics.

    Original languageEnglish
    Article numbere17625
    Pages (from-to)-
    Number of pages17
    JournalPLoS ONE
    Volume6
    Issue number3
    DOIs
    Publication statusPublished - 22 Mar 2011

    Keywords

    • GENE-EXPRESSION PROFILES
    • FINE-NEEDLE-ASPIRATION
    • BREAST-CANCER
    • ENDOTHELIAL FUNCTION
    • NANOGRAM AMOUNTS
    • SINGLE-CELL
    • MICROARRAY
    • AMPLIFICATION
    • SAMPLES
    • REPRODUCIBILITY

    Cite this

    Kennedy, Laura ; Pauriah, Maheshwar ; Godfrey, Valerie ; Howie, Jacqueline ; Dennis, Helen ; Crowther, Daniel ; Struthers, Allan ; Goddard, Catharine ; Feuerstein, Giora ; Lang, Chim ; Miele, Gino. / Global Array-Based Transcriptomics from Minimal Input RNA Utilising an Optimal RNA Isolation Process Combined with SPIA cDNA Probes. In: PLoS ONE. 2011 ; Vol. 6, No. 3. pp. -.
    @article{0f858af7d24b49638c2b7aa63672664f,
    title = "Global Array-Based Transcriptomics from Minimal Input RNA Utilising an Optimal RNA Isolation Process Combined with SPIA cDNA Probes",
    abstract = "Background: Technical advances in the collection of clinical material, such as laser capture microdissection and cell sorting, provide the advantage of yielding more refined and homogenous populations of cells. However, these attractive advantages are counter balanced by the significant difficultly in obtaining adequate nucleic acid yields to allow transcriptomic analyses. Established technologies are available to carry out global transcriptomics using nanograms of input RNA, however, many clinical samples of low cell content would be expected to yield RNA within the picogram range. To fully exploit these clinical samples the challenge of isolating adequate RNA yield directly and generating sufficient microarray probes for global transcriptional profiling from this low level RNA input has been addressed in the current report. We have established an optimised RNA isolation workflow specifically designed to yield maximal RNA from minimal cell numbers. This procedure obtained RNA yield sufficient for carrying out global transcriptional profiling from vascular endothelial cell biopsies, clinical material not previously amenable to global transcriptomic approaches. In addition, by assessing the performance of two linear isothermal probe generation methods at decreasing input levels of good quality RNA we demonstrated robust detection of a class of low abundance transcripts (GPCRs) at input levels within the picogram range, a lower level of RNA input (50 pg) than previously reported for global transcriptional profiling and report the ability to interrogate the transcriptome from only 10 pg of input RNA. By exploiting an optimal RNA isolation workflow specifically for samples of low cell content, and linear isothermal RNA amplification methods for low level RNA input we were able to perform global transcriptomics on valuable and potentially informative clinically derived vascular endothelial biopsies here for the first time. These workflows provide the ability to robustly exploit ever more common clinical samples yielding extremely low cell numbers and RNA yields for global transcriptomics.",
    keywords = "GENE-EXPRESSION PROFILES, FINE-NEEDLE-ASPIRATION, BREAST-CANCER, ENDOTHELIAL FUNCTION, NANOGRAM AMOUNTS, SINGLE-CELL, MICROARRAY, AMPLIFICATION, SAMPLES, REPRODUCIBILITY",
    author = "Laura Kennedy and Maheshwar Pauriah and Valerie Godfrey and Jacqueline Howie and Helen Dennis and Daniel Crowther and Allan Struthers and Catharine Goddard and Giora Feuerstein and Chim Lang and Gino Miele",
    year = "2011",
    month = "3",
    day = "22",
    doi = "10.1371/journal.pone.0017625",
    language = "English",
    volume = "6",
    pages = "--",
    journal = "PLoS ONE",
    issn = "1932-6203",
    publisher = "Public Library of Science",
    number = "3",

    }

    Global Array-Based Transcriptomics from Minimal Input RNA Utilising an Optimal RNA Isolation Process Combined with SPIA cDNA Probes. / Kennedy, Laura; Pauriah, Maheshwar; Godfrey, Valerie; Howie, Jacqueline; Dennis, Helen; Crowther, Daniel; Struthers, Allan; Goddard, Catharine; Feuerstein, Giora; Lang, Chim; Miele, Gino.

    In: PLoS ONE, Vol. 6, No. 3, e17625, 22.03.2011, p. -.

    Research output: Contribution to journalArticle

    TY - JOUR

    T1 - Global Array-Based Transcriptomics from Minimal Input RNA Utilising an Optimal RNA Isolation Process Combined with SPIA cDNA Probes

    AU - Kennedy, Laura

    AU - Pauriah, Maheshwar

    AU - Godfrey, Valerie

    AU - Howie, Jacqueline

    AU - Dennis, Helen

    AU - Crowther, Daniel

    AU - Struthers, Allan

    AU - Goddard, Catharine

    AU - Feuerstein, Giora

    AU - Lang, Chim

    AU - Miele, Gino

    PY - 2011/3/22

    Y1 - 2011/3/22

    N2 - Background: Technical advances in the collection of clinical material, such as laser capture microdissection and cell sorting, provide the advantage of yielding more refined and homogenous populations of cells. However, these attractive advantages are counter balanced by the significant difficultly in obtaining adequate nucleic acid yields to allow transcriptomic analyses. Established technologies are available to carry out global transcriptomics using nanograms of input RNA, however, many clinical samples of low cell content would be expected to yield RNA within the picogram range. To fully exploit these clinical samples the challenge of isolating adequate RNA yield directly and generating sufficient microarray probes for global transcriptional profiling from this low level RNA input has been addressed in the current report. We have established an optimised RNA isolation workflow specifically designed to yield maximal RNA from minimal cell numbers. This procedure obtained RNA yield sufficient for carrying out global transcriptional profiling from vascular endothelial cell biopsies, clinical material not previously amenable to global transcriptomic approaches. In addition, by assessing the performance of two linear isothermal probe generation methods at decreasing input levels of good quality RNA we demonstrated robust detection of a class of low abundance transcripts (GPCRs) at input levels within the picogram range, a lower level of RNA input (50 pg) than previously reported for global transcriptional profiling and report the ability to interrogate the transcriptome from only 10 pg of input RNA. By exploiting an optimal RNA isolation workflow specifically for samples of low cell content, and linear isothermal RNA amplification methods for low level RNA input we were able to perform global transcriptomics on valuable and potentially informative clinically derived vascular endothelial biopsies here for the first time. These workflows provide the ability to robustly exploit ever more common clinical samples yielding extremely low cell numbers and RNA yields for global transcriptomics.

    AB - Background: Technical advances in the collection of clinical material, such as laser capture microdissection and cell sorting, provide the advantage of yielding more refined and homogenous populations of cells. However, these attractive advantages are counter balanced by the significant difficultly in obtaining adequate nucleic acid yields to allow transcriptomic analyses. Established technologies are available to carry out global transcriptomics using nanograms of input RNA, however, many clinical samples of low cell content would be expected to yield RNA within the picogram range. To fully exploit these clinical samples the challenge of isolating adequate RNA yield directly and generating sufficient microarray probes for global transcriptional profiling from this low level RNA input has been addressed in the current report. We have established an optimised RNA isolation workflow specifically designed to yield maximal RNA from minimal cell numbers. This procedure obtained RNA yield sufficient for carrying out global transcriptional profiling from vascular endothelial cell biopsies, clinical material not previously amenable to global transcriptomic approaches. In addition, by assessing the performance of two linear isothermal probe generation methods at decreasing input levels of good quality RNA we demonstrated robust detection of a class of low abundance transcripts (GPCRs) at input levels within the picogram range, a lower level of RNA input (50 pg) than previously reported for global transcriptional profiling and report the ability to interrogate the transcriptome from only 10 pg of input RNA. By exploiting an optimal RNA isolation workflow specifically for samples of low cell content, and linear isothermal RNA amplification methods for low level RNA input we were able to perform global transcriptomics on valuable and potentially informative clinically derived vascular endothelial biopsies here for the first time. These workflows provide the ability to robustly exploit ever more common clinical samples yielding extremely low cell numbers and RNA yields for global transcriptomics.

    KW - GENE-EXPRESSION PROFILES

    KW - FINE-NEEDLE-ASPIRATION

    KW - BREAST-CANCER

    KW - ENDOTHELIAL FUNCTION

    KW - NANOGRAM AMOUNTS

    KW - SINGLE-CELL

    KW - MICROARRAY

    KW - AMPLIFICATION

    KW - SAMPLES

    KW - REPRODUCIBILITY

    U2 - 10.1371/journal.pone.0017625

    DO - 10.1371/journal.pone.0017625

    M3 - Article

    VL - 6

    SP - -

    JO - PLoS ONE

    JF - PLoS ONE

    SN - 1932-6203

    IS - 3

    M1 - e17625

    ER -