Abstract
Reliable extraction and sensitive detection of RNA from human peripheral blood mononuclear cells (PBMCs) is critical for a broad spectrum of immunology research and clinical diagnostics. RNA analysis platforms are dependent upon high-quality and high-quantity RNA; however, sensitive detection of specific responses associated with high-quality RNA extractions from human samples with limited PBMCs can be challenging. Furthermore, the comparative sensitivity between RNA quantification and best-practice protein quantification is poorly defined. Therefore, we provide herein a critical evaluation of the wide variety of current generation of RNA-based kits for PBMCs, representative of several strategies designed to maximize sensitivity. We assess these kits with a reverse transcription quantitative PCR (RT-qPCR) assay optimized for both analytically and diagnostically sensitive cell-based RNA-based applications. Specifically, three RNA extraction kits, one post-extraction RNA purification/concentration kit, four SYBR master-mix kits, and four reverse transcription kits were tested. RNA extraction and RT-qPCR reaction efficiency were evaluated with commonly used reference and cytokine genes. Significant variation in RNA expression of reference genes was apparent, and absolute quantification based on cell number was established as an effective RT-qPCR normalization strategy. We defined an optimized RNA extraction and RT-qPCR protocol with an analytical sensitivity capable of single cell RNA detection. The diagnostic sensitivity of this assay was sufficient to show a CD8+ T cell peptide epitope hierarchy with as few as 1 × 104 cells. Finally, we compared our optimized RNA extraction and RT-qPCR protocol with current best-practice immune assays and demonstrated that our assay is a sensitive alternative to protein-based assays for peptide-specific responses, especially with limited PBMCs number. This protocol with high analytical and diagnostic sensitivity has broad applicability for both primary research and clinical practice.
Highlights
Reliable isolation of high quality and high quantity RNA from peripheral blood mononuclear cells (PBMCs) and other cells is critical for a broad range of basic, preclinical, and clinical applications [1, 2]
Four master-mixes—ssoAdvancedTM Universal SYBR R Green Master-Mix (Bio-Rad), QuantiNova SYBR R Green PCR Kit (QIAGEN), PowerUp SYBR R Green Master-Mix (Applied Biosystems) and RT2 SYBR R Green qPCR Master-Mix (QIAGEN)—were evaluated using two methods of preparing reference standards: (i) standards derived from log10 diluted amplicon; and (ii) standards generated from log10 diluted cDNA (Figure 1A)
We describe an optimized RNA extraction and reverse transcription quantitative PCR (RT-qPCR) protocol requiring low PBMC numbers, with high analytical and diagnostic sensitivity, whilst maintaining high correlation to protein-level quantification that is typically reliant on much larger cell numbers for detection
Summary
Reliable isolation of high quality and high quantity RNA from peripheral blood mononuclear cells (PBMCs) and other cells is critical for a broad range of basic, preclinical, and clinical applications [1, 2]. An alternative is absolute quantification normalized to cell number, which minimizes this potential analytical bias [48,49,50] To address this need, we developed a highly sensitive RNA extraction and RT-qPCR quantification strategy for analysis of gene expression from human PBMCs. We compared the efficiency of the latest generation of SYBR mastermixes and RNA extraction and reverse transcription kits, taking into consideration both total RNA yield and RNA concentration. Kits were evaluated using RNA extracted using the MagMAXTM mirVanaTM Total RNA Isolation Kit (MagMAX) with or without the RNeasy R MiniElute Cleanup Kit. Briefly, 1 × 106 PBMCs were incubated for 6 h with or without PMA/Iono as described above.
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