An Analytically and Diagnostically Sensitive RNA Extraction and RT-qPCR Protocol for Peripheral Blood Mononuclear Cells

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.


INTRODUCTION
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). RNA-based assays enable analysis of basal expression profiles and responses to antigen or mitogen stimulation (3,4). Human PBMCs are a common source of RNA as collection of blood is less invasive and allows in-depth monitoring of many aspects of immunobiology (1,5) including identification, classification and prognosis of cancers (6)(7)(8)(9)(10)(11) monitoring inflammation (12,13), and evaluating therapeutic efficacy (14)(15)(16)(17).
A range of RNA-based platforms are now available, all dependent upon high quality and high quantity RNA (1). However, an important requirement for many applications is both excellent analytical sensitivity (i.e., smallest number of cells detectable) and diagnostic sensitivity (i.e., smallest detectable response to stimulation) (18). Protein-level immunoassays (e.g., flow cytometry, cytokine bead-based arrays, ELIspot) (19)(20)(21)(22) are routinely used to detect PBMCs response to stimulation (23)(24)(25). Indeed, ELIspot has been used extensively as the "gold standard" immune assay given its sensitivity and has been optimized and validated as part of the global HIV/AIDS Comprehensive T Cell Vaccine Immune Monitoring Consortium (26)(27)(28). However, these protocols are limited by the relatively high number of cells required, especially when considering targets with low frequencies (24), when collection of large blood volumes is challenging (29,30), or when there are many experimental variables [e.g., vaccine/peptide (14,17,31,32) or epitope testing (33)(34)(35)]. Therefore, there is an unmet need for a robust RNA extraction and transcriptomic analysis protocol from limited input cell numbers (e.g., PBMCs) with high analytical and diagnostic sensitivity that meets or exceeds that of proteinlevel immuno-assays.
Reverse transcription quantitative PCR (RT-qPCR) remains the "Gold Standard" for assay of gene expression as an alternate readout to protein expression (36,37). RT-qPCR is more sensitive than traditional RNA quantification technologies (i.e., Northern blotting, nuclease protection assays, in-situ hybridization, RNA microarrays etc.) (38)(39)(40). More recent technologies such as Sanger and next-generation sequencing (i.e., RNA-Seq, single cell RNA-seq, NanoString) and advanced PCR methods (i.e., digital PCR) are similarly sensitive (41,42) but are relatively expensive or further require complex bioinformatical analysis (43,44). In contrast, our optimized RT-qPCR assay is designed specifically for cheap, robust, reproducible and sensitive analysis of gene expression, is available to almost any laboratory, and serves as a sensitive and specific alternative to protein expression. Additionally, by focusing on a limited number of genes, RT-qPCR is ideal for validation of genes of interest identified from more untargeted methods such as RNAseq.
However, there is an unmet need for a robust RNA extraction and RT-qPCR protocol with excellent analytical and diagnostic sensitivity, ideally to the single cell level. An important consideration for such a protocol is that RT-qPCR normalization can be achieved by either absolute quantification of copies per reaction using a standard curve, or by semi-quantitative foldchange of relative expression normalized to reference genes (39,45). However, in vitro stimulation has been shown to modulate the expression of many commonly used reference genes (46,47), and key assumptions underlying semi-quantitative analysis require consistent reference gene expression across experimental conditions within and amongst cell populations. 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. We determined that ssoAdvanced TM Universal SYBR R Green Master-Mix provided optimal reaction efficiency, whilst SuperScript TM IV Reverse Transcriptase had the highest cDNA yields. We demonstrated significantly increased PBMC RNA recovery following extraction with the magnetic bead-based MagMAX TM mirVana TM kit, with no further enhancement of analytical sensitivity by including an additional step of RNA concentration. When testing the analytical sensitivity of our optimized protocol, we could detect RNA to the single cell level of highly expressed genes. Furthermore, by evaluating a hierarchy of CD8 + T cell epitope responses, we demonstrated diagnostic sensitivity with as few as 1 × 10 4 PBMCs. This optimized RNA extraction and RT-qPCR protocol, with high analytical and diagnostic sensitivity, provides a robust alternative to proteinbased immune assays.  Table 1).

PBMCs
Blood was collected from healthy donors or buffy coats (n = 12) provided by the Australian Red Cross Blood Service, under a protocol approved by the James Cook University Human Research Ethics Committee (#H6702). PBMCs were isolated by density gradient centrifugation and cryopreserved in FBS 10% DMSO. Prior to use, samples were thawed rapidly at 37 • C, treated with DNAase I (1 µg/mL; StemCell), and rested for 18 h at 2 × 10 6 cells/mL in media (RPMI-1640, 10% FBS, 100 U/mL penicillin/streptomycin) at 37 • C and 5% CO 2 . Viable PBMCs were counted prior to downstream analysis.

HLA Typing
Genomic DNA was isolated from PBMCs using the QIAamp DNA Mini Kit (QIAGEN) according to manufacturer's instructions. High-resolution class I and class II HLA typing was performed by the Australian Red Cross Transplant and Immunological Services (Melbourne, Australia) using the MIA FORA NGS FLEX HLA typing kit (Immunocor) and Illumina MiSeq and MiniSeq platforms.

Quantitative PCR
Assay Setup qPCR was conducted using the QuantStudio 3 Real-Time PCR system running QuantStudio Design and Analysis Software  (45).

SYBR Master-Mix Testing: cDNA Standard
The four SYBR master-mix kits were further evaluated with efficiency titrations of cDNA standards. Briefly, RNA was extracted from 1 × 10 6 unstimulated PBMCs using the RNeasy R Mini Kit (QIAGEN). Seven microliters of extracted RNA was converted to cDNA using the SuperScript TM III First-Strand Synthesis System kit (Invitrogen). Master-mix reaction efficiency was calculated from log 10 diluted cDNA (10 4 -10 1 cells/reaction) with RPL13a, SDHA, TBP, and IFN-γ primers at 500 nM.
Reference Gene Stability Testing 1 × 10 6 PBMCs were stimulated for 6, 12, 16, 24, or 48 h with or without PMA/Iono as described above. RNA was extracted with RNeasy R Mini Kit (QIAGEN). Seven microliters of extracted RNA was reverse transcribed with SuperScript TM III First-Strand Synthesis System kit (Invitrogen). qPCR was run with ssoAdvanced TM master-mix, RPL13a, SDHA, TBP and IFN-γ primers at 500 nM and samples at 10 2 cells/reaction.

Evaluation of RNA Extraction Kits
To evaluate RNA yield and quality, three RNA extraction kits: . qPCR was run with ssoAdvanced TM mastermix, RPL13a, SDHA, TBP, and IFN-γ primers at 500 nM and sample diluted to 10 2 cells/reaction; except when considering concentration, when the samples were run undiluted.

Analytical and Diagnostic Sensitivity
For determination of analytical sensitivity, RNA was extracted from a log 10 serial dilution of unstimulated PBMCs (10 6 -10 0 cells/extraction), using the MagMAX kit with or without the RNeasy R MiniElute Cleanup Kit. A media-only extraction control was processed in parallel. For determination of diagnostic sensitivity, RNA was extracted using the MagMAX kit from titrated PBMCs (4 × 10 5 , 1 × 10 5 , 2.5 × 10 4 , and 1 × 10 4 ) incubated for 6 h with or without PMA/Iono or HLAmatched peptide. For sensitivity evaluations, 10 µL of RNA was converted to cDNA using the SuperScript TM IV First-Strand Synthesis System (Invitrogen). qPCR used undiluted sample with ssoAdvanced TM master-mix, RPL13a, SDHA, TBP, and IFN-γ primers at 500 nM.

Data Analysis
RT-qPCR, Bioanalyzer and NanoDrop data were analyzed using a repeated-measures two-way ANOVA with Bonferroni-corrected multiple comparisons test comparing test to control mean. Correlation between RT-qPCR and protein quantification was test with linear regression analysis. Analysis was conducted using GraphPad Prism version 7.0 (GraphPad). In all cases, P < 0.05 were considered significant.

ssoAdvanced TM Universal SYBR ® Green Master-Mix Provided the Highest Reaction Efficiency
Four master-mixes-ssoAdvanced TM 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 log 10 diluted amplicon; and (ii) standards generated from log 10 diluted cDNA ( Figure 1A). Reaction efficiency was quantified using four primer sets: three sets targeted reference genes known to have high (60S ribosomal protein L13a; RPL13a), moderate (Succinate dehydrogenase complex, subunit A; SDHA) and low (TATAbinding protein; TBP) expression; and one set targeted a cytokine gene (interferon gamma; IFN-γ ) (47). When considering an acceptable reaction efficiency range (90-110%), 35.4% of the amplicon-derived standards ( Mitogen Stimulation Induced Changes in RPL13a, SDHA, and TBP Gene Expression The expression stability of three commonly used reference genes (47,53,54), RPL13a, SDHA, and TBP, previously reported as stable in PBMCs following stimulation (47), were evaluated by RT-qPCR within PBMCs stimulated with PMA/Iono for 6, 12, 18, 24, or 48 h. Expression of all three genes changed over time with cell culture, and significantly increased at 48 h post-stimulation as compared to baseline (P < 0.001, P < 0.01, and P < 0.01, respectively; Figure 1B). These data establish that the expression of common reference genes is significantly affected by stimulation, emphasizing the importance of absolute quantification normalized to cell numbers, rather than relative quantification.

Magnetic Bead-Based Extraction Significantly Increased RNA Yield and Concentration
Next, RNeasy R Mini and Micro silica columns (both QIAGEN) and MagMAX TM mirVana TM (MagMAX) Total RNA Isolation (Applied Biosystems) kits were tested for 1) RNA yield and 2) concentration with or without a post-extraction RNA concentration step using the RNeasy R MiniElute Cleanup Kit (QIAGEN). In each case, PBMCs were incubated with or without PMA/Iono for 6 h. RIN assessment demonstrated that RNA integrity was high (>7) and consistent across all kits (Figure 2A

Single Cell Analytical Sensitivity Was Observed Following Magnetic Bead-Based RNA Extraction
We next evaluated the analytical sensitivity of our optimized protocol using MagMAX extraction kit ( Figure 4A) and MagMAX-RNeasy R MiniElute extraction-concentration kit ( Figure 4B). RNA was extracted from a log 10 serial dilution of unstimulated PBMCs and expression of IFN-γ , RPL13a, SDHA, and TBP determined by absolute quantification. The highly expressed RPL13a gene was detected at single-cell level from both MagMAX (0.88 log 10 copies/reaction; Figure 4A) and MagMAX-RNeasy R MiniElute combination (0.90 log 10 copies/reaction; Figure 4B) extractions. Extraction technique did not influence IFN-γ , RPL13a, or TBP expression; whilst variability in SDHA expression (P Ext < 0.05) was driven by increased RT-qPCR signal at 10 5 and 10 4 PBMCs per extraction. These data establish that our optimized protocol is capable of extracting and quantifying RNA of a highly expressed gene from a single cell. Importantly, the additional step of RNeasy R MiniElute Cleanup did not further enhance analytical sensitivity.

RT-qPCR Protocol Diagnostic Sensitivity Correlates Significantly With Protein Level
Quantification of Epitope-Specific Stimulation From as Few as 1 × 10 4 PBMCs Next, we determined the diagnostic sensitivity of our optimized RNA extraction and RT-qPCR protocol to confirm that it accurately reflected data generated using protein-level assays. We evaluated the epitope-specific stimulatory response for four CD8 + T cell epitopes restricted by different MHC molecules, quantifying IFN-γ production by flow cytometry, cytokine bead array and ELIspot; and IFN-γ mRNA by our optimized protocol. A limited epitope-specific IFN-γ response was demonstrated by flow cytometry (Figure 5A) and bead arrays ( Figure 5B) whereas all samples were observed to respond to stimulation by ELIspot ( Figure 5C). Importantly, our RT-qPCR protocol (Figure 6), was able to replicate the ELIspot results but with significantly reduced cell numbers (HLA-A1 2.5 × 10 4 , -A2 1 × 10 5 , -B7 1 × 10 4 , -B8 1 × 10 5 ; 48-fold, 12-fold, and 48-fold and 12-fold, respectively; Figure 5D).

Optimized RT-qPCR Assay Correlated With Best-Practice Protein-Based Immunoassays
Next, we assessed correlation between results obtained using our optimized RT-qPCR protocol and current best-practice immunoassays. The correlation between our RT-qPCR protocol and ELIspot was significant at all cell numbers tested [P = 0.0006, R 2 = 0.7063 (4 ×  (Figure 5E, left panel). Thus, data generated using our optimized RT-qPCR assay are consistent with best practice protein-based immunoassays. Furthermore, our assay is capable of defining an epitope-specific response hierarchy from as few as 1 × 10 4 cells, representing a clinically and diagnostically meaningful reduction in cell number. Taken together, we report here a highly sensitive RNA extraction and RT-qPCR quantification strategy using the MagMAX RNA extraction kit, Superscript TM IV reversetranscription kit and ssoAdvanced TM SYBR master-mix (Supplementary Table 3). This assay is sensitive to the single cell level, can define an epitope hierarchy of response from as few as 1 × 10 4 cells, and represents a sensitive and robust alternative to protein quantification for research, diagnostic and clinical applications.

DISCUSSION
Herein, we describe an optimized RNA extraction and 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.
Precise RT-qPCR results are typically dependent on reactions maintaining efficiency close to 100% (45). Both assay design (e.g., primer concentration, master-mix) and sample (e.g., , n = 12 total) were stimulated with synthetic HLA-matched peptides representing CMV, Influenza or EBV CD8 + T cell epitopes "VTE," "GIL," "RPH," or "FLR" (black), respectively. All samples were cultured with media negative control or PMA/Iono positive control. IFN-γ mRNA expression was determined by absolute quantification RT-qPCR of titrated PBMCs (4 × 10 5 , 1 × 10 5 , 2.5 × 10 4 , and 1 × 10 4 ); gene copy number per reaction was quantified by standard curve and log 10 transformed. Single RNA extractions, with single reverse transcription reactions per n, were performed. qPCR performed in technical triplicate replicates. Flow cytometry and MagPIX performed in single wells. ELIspot performed in technical triplicate replicates. Sample mean calculated from the mean of the technical single or triplicates. Biological mean ± technical SEM above background shown. co-extracted inhibitors) may influence PCR efficiency. We made use of the open-access database PrimerBank TM since those primers have been designed for use under consistent conditions (i.e., optimal Tm 60 • C) and cover most known human and mouse genes (55). We found primer concentration titrations did not impact reaction efficiency, whereas the SYBR master-mix had a significant impact. PCR inhibitors, including hemoglobin, lactoferrin, anticoagulants, IgG, polysaccharides, and proteases, can be co-extracted in PBMC preparations (56,57). It is known that some DNA-polymerase variants and PCR buffer "enhancers" have improved reaction efficiency in the presence of such inhibitors (56,58). The ssoAdvanced TM master-mix, identified herein as optimal of those tested, appears to be one such master-mix facilitating PCR efficiency in the presence of co-extracted inhibitors. Optimization of mastermix reagents will likely continue to be important in improving blood-based PCR analysis and diagnostics (59,60); especially for accurate amplification of relatively low abundant targets, comparisons between populations with high variability, or amplification from inhibitor-enriched mediums (i.e., whole blood extractions) (10,59). We tested three RNA extraction kits by evaluating extraction quality and efficiency: RNeasy R Mini Kit, RNeasy R Micro Kit, and MagMAX TM mirVana TM Kit; in combination with the RNA purification and concentration kit: RNeasy R MiniElute Cleanup Kit. When extracting identically controlled samples, all kits yielded RNA with equivalent RIN scores and low technical variability between replicates. Importantly, RNA yield from PBMCs was significantly increased using MagMAX as compared to silica-column technologies. Additionally, when compared to silica-column extractions, we found MagMAX was more cost and time efficient when running larger number of samples (e.g., 96 samples in ∼2 h). We therefore expect magnetic bead-based extractions will become increasingly common within bloodbased nucleic acid isolations (59,61,62). In addition to extraction techniques (e.g., silica column, phase separation), other factors that could impact RNA quality, yield and concentration include sample collection, storage, and transportation.
Four reverse transcriptase (RT) kits were also evaluated: SuperScript TM III First-Strand Synthesis System, SuperScript TM IV First-Strand Synthesis System, iScript TM Advanced cDNA Synthesis Kit and High-Capacity RNA-cDNA Kit TM . Of those, Superscript TM IV was associated with the highest qPCR signal. A previous study evaluating RNA extracted from PBMCs using earlier-generation RT kits reported >128-fold increased qPCR signal between kits (1). We speculate that the reduced variability that we observed between RT kits tested in our study reflects consistent kit quality, purity of the RNA extracted by MagMAX, or a combination thereof.
Both analytical sensitivity and diagnostic sensitivity are key criteria for any RT-qPCR protocol. We show that the analytical sensitivity of our assay is to the level of single cell RNA detection for relatively highly expressed RPL13a. Sample concentration and clean-up has been suggested to remove inhibitors and increase sensitivity (63,64). Unexpectedly, we found this step did not improve our analytical sensitivity, and was timeconsuming, expensive and reduced sample volume. Nevertheless, if concentration is warranted under specific experimental situations, our data suggest that it is technically feasible while retaining high analytical sensitivity. Diagnostic sensitivity determined using MagMAX showed that an epitope response hierarchy could be detected with as few as 1 × 10 4 PBMCs. It is well-known that there is no absolute correlation between RNA expression and protein translation. Indeed, correlations between transcript and protein expression would be markedly reduced under situations of epigenetic, post-transcriptional or post-translational modification of the gene of interest (65). Nevertheless, we correlated our optimized RT-qPCR assay with commonly used protein-level immunoassays (flow cytometry, cytokine bead arrays, and ELIspot) and showed a very high correlation with the gold-standard protein-level assay, ELIspot, as well as the commonly used MagPIX bead-based cytokine assay, at all tested PBMC concentrations. This highlights that our protocol represents a robust alternative to protein-based assays (e.g., when measuring changes in cytokine mRNA expression in PBMCs in response to specific in vitro stimulation). This work will significantly improve analytical capacity of studies relying on irreplaceable, relatively small or costly human samples (e.g., neonatal PBMCs) (66,67).
Another important outcome of our work is the finding that absolute quantification of transcripts and subsequent normalization to cell numbers is the most appropriate analysis strategy for RNA/RT-qPCR quantification from PBMCs (45,48). We observed significant alterations in gene expression of commonly used reference genes RPL13a, SDHA and TBP following stimulation. This is not unexpected as reference genes have been described as variable across cell types, tissues, and experimental and stimulatory conditions (25,47,68,69).
In summary, we report herein the development of an optimized PBMC RNA extraction and RT-qPCR protocol. We employed a qPCR strategy of absolute quantification utilizing PrimerBank TM primers and ssoAdvanced TM Universal SYBR R Green Master-Mix. PBMC RNA was isolated with MagMAX TM mirVana TM Total RNA Isolation Kit and reverse transcribed with SuperScript TM IV First-Strand Synthesis System. Our assay provided single cell analytical sensitivity and a diagnostic sensitivity that could define response hierarchy from 1 × 10 4 cells. This assay offers an alternative to current best practice protein-based immunoassays, especially for limited PBMC numbers. This work has broad applicability for both clinical and primary research practice.

DATA AVAILABILITY STATEMENT
The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation, to any qualified researcher.

ETHICS STATEMENT
The studies involving human participants were reviewed and approved by James Cook University Human Research Ethics Committee. The patients/participants provided their written informed consent to participate in this study.

ACKNOWLEDGMENTS
We are grateful to all of the donors who kindly provided samples in support of this study. Figure 6 was created using BioRender.com.