Whole venous blood was collected from healthy volunteers and either 2.5, 1.0 or 0.3 mL added to the Paxgene blood RNA reagent in the same ratio as the manufacturers guidelines. The total RNA yield from 2.5, 1.0 and 0.3 mL of whole blood was 4.5 – 11.6 μg, 5.1 – 8.3 μg and 1.6 to 5.0 μg respectively. With optical density ratios (260/280) of 1.97–2.14. Figure 1 shows Agilent 2100 Bioanalyser traces for the total RNA extracted from the different volumes of whole blood. Panel A shows the full scale (2.5 mL) blood RNA extraction, panels B and C show total RNA from the low volume extractions, for comparison panel D shows the trace for Stratagene Universal RNA. The 18s and 28s RNA peaks can be seen at approximately 42 and 49 seconds respectively. Both the 2.5 mL and 1.0 mL extractions were loaded on to Eukaryote total RNA Nano chips whereas the 0.3 mL extractions were loaded on to Eukaryote total RNA Pico chips. The Bioanalyser software generates RNA Integrity Numbers (RIN) for each sample giving an estimate of the RNA integrity. Total RNA extracted from 0.3 – 2.5 mL had a RIN of between 8.2 and 9.6. RNA was reverse transcribed and assayed for a panel of reference genes to ensure expression profiles were maintained across all volumes extracted.

Whole venous blood samples were collected from 87 children with confirmed invasive pneumococcal disease. A total of 48 children (55%) were male, and the age range was 0.17 to 13 years, median age 3.25 years. There were 25 (28.7%) non-survivors. Of the children with IPD, 75 had meningitis(86%) and 12 had pneumonia (14%).

Total RNA yield from 0.3 mL of whole blood varied between 1.1 and 2.9 μg with 260/280 ratios of 1.91 – 2.03. Eight small volume blood samples were processed using the method described below and amplified for the 3 GAPDH assays. Figure 2a shows that in all cases a good signal is detected across the whole GAPDH gene, thereby indicating that full length mRNA has been isolated. Figure 2b shows the raw Ct values obtained for the 8 samples with a panel of 7 additional housekeeper genes. The samples show excellent consistency in the expression levels for all the housekeeper assays further indicating that all the RNA samples have good mRNA integrity. The RT qPCR assays were shown to be a more sensitive method of RNA QC analysis for samples and could be used in place of the Bioanalyser.

The RNA yields were low but sufficient cDNA was produced to perform RT qPCR experiments to quantify ICAM and IL-6 gene expression. Relative gene expression (2^-ΔΔCt) was significantly higher in survivors compared to non-survivors and controls (ICAM: p < 0.0005 and IL-6: p = 0.003, Kruskal Wallis). Relative gene expression was significantly lower than in non-survivors than survivors; ICAM: median (IQR); 3.56 (0.82 – 5.72) versus 4.41 (1.44 – 9.57), and IL-6: median (IQR); 2.16 (0.71 – 5.72) versus 6.73 (1.17 – 14.93). Relative gene expression was significantly lower in controls than cases ICAM: 1.00 (0.54 – 1.79) versus 4.17(1.29 – 8.06) and IL-6: 0.92 (0.49 – 2.28) versus 4.32(0.81 – 13.27) (p < 0.0005 and p = 0.01 respectively) (Figure 3).

Children in this study were admitted to the Queen Elizabeth Central Hospital, Blantyre, Malawi, and recruited into a prospective observational study of host determinants of invasive pneumococcal disease susceptibility and severity in Malawian children, the details of which have been described elsewhere [18]. Controls were taken from healthy, afebrile children from the same villages as the cases. The study protocol was approved by the College of Medicine Research Ethics Committees, both in Blantyre, Malawi and the Local Research Ethics Committee at the Liverpool School of Tropical Medicine, United Kingdom. Written informed consent was obtained from parents or guardians before children were included into the study.

Peripheral whole blood (0.3 mL) from venepuncture was dispensed into micro-tubes pre-aliquoted with PAXgene™ reagent (0.86 mL), keeping the blood:reagent ratio the same as in the PAXgene™ Blood RNA Tubes [Qiagen]. The sample was gently inverted and stored at -80°C within two hours of collection.

RNA was extracted from whole blood using the PAXgene™ Blood RNA System Kit employing an amended version of the manufacturer's guidelines. Briefly, the samples were removed from -80°C and incubated at room temperature for 2 hours to ensure complete lysis. Following lysis the tubes were centrifuged for 10 min at 5,000 × g (Boeco M-24 centrifuge), the supernatant decanted and 500 μL of RNase-free water added to the pellet. The tube was vortexed to thoroughly re-suspend the pellet, centrifuged for 10 min at 5000 × g and the entire supernatant discarded. The remaining pelleted lysate was re-suspended in 360 μL of buffer BR1 by vortexing and the manufacturer's protocol was followed from this step.

Freshly extracted RNA was measured using a NanoDrop ND-1000 UV-visible spectrophotometer [Labtech International, Ringmer, UK]. The software displays the concentration in ng/μL. There is also a quality output, which provides 260/280 and 260/230 ratios enabling purity estimations. RNA integrity was additionally assessed using the Agilent 2100 Bioanalyser [Agilent Technologies]. Samples were loaded on to either the Eukaryote total RNA nano chip or the Eukaryote total RNA pico chip.

To evaluate the utility of the RNA achieved by this method we then carried out RT qPCR analysis on a panel of 10 assays on a total of 8 housekeeper genes as detailed in Table 1. As a further quality control (QC) measure with enhanced sensitivity, we developed assays to 3 regions of GAPDH; termed 3', mid and 5'. By using oligo dT for the priming step during reverse transcription it is possible to use the presence of the mid and 5' assays for GAPDH to confirm that full length transcripts are present. RNA (1 μg) was reverse transcribed using SuperScript II™ reverse transcriptase (Invitrogen Ltd, Paisley, UK) following the manufacturer's guidelines, with oligo dT_12–18 used for priming. RT qPCR assays were designed using locked nucleic acid analogues (LNA) of fluorescence resonance energy transfer hydrolysis probes [19] employed in the Human Universal Probe Library system (Roche, Switzerland), with amplification primers obtained from MWG (Edersberg, Germany). Experiments were performed on an ABI 7900 Real-Time Sequence Detection System in 384 well format, using an EpMotion 5070 robot (Eppendorf, Germany) for plate set up.

RNA was reverse transcribed using SuperScript II™ RNase H reverse transcriptase (Invitrogen Ltd, Paisley, UK) following the manufacturer's guidelines. Reactions were primed using 1 μL oligo (dT)_12–18 (500 μg/mL) and took place in the presence of RNaseOUT™ (all reagents were from Invitrogen Ltd, Paisley, UK)

The Human Universal Probe Library system [19] (Roche, Switzerland) employing proprietary LNA analogues was used for RT qPCR to measure expression levels in genes of interest. Using the Roche Online Assay Design Centre, specific primers and an associated probe were selected for the ICAM transcript. To compensate for variations in cell number, RNA isolation, reverse transcription and PCR amplification efficiency, three endogenous 'house-keeping' transcripts were chosen using the GeNORM algorithm [20]. Briefly, the cases and controls were screened for 8 reference genes, the algorithm works out the stability of each transcript, and sequentially removes the least stable transcript until the three most stable transcripts remain. These were; HPRT1 (Hypoxanthine phosphoribosyl transferase 1), YWHAZ (tyrosine 3 monooxygenase) and GAPDH glyceraldehyde 3 phosphate dehydrogenase.

cDNA was diluted 1 in 50. Each reaction comprised of 5 μL diluted cDNA, 10 μL 2× qPCR Master Mix with UNG (Eurogentec Ltd., Southampton, UK), 0.2 μL each primer (20 μM) (Metabion, Planegg-Martinsried, Germany), 0.2 μL Probe (10 μM) Human Universal Probe Library system [19] (Roche, Switzerland), and 4.4 μL of water. Samples were then amplified on an ABI 7700 PRISM SDS, and the temperature programme resembled 50°C for 2 min, 95°C for 10 min followed by 40 cycles of 95°C for 15 sec and 60°C for 1 min. The amounts of target genes expressed in a sample are normalized to the average of the three endogenous controls. This is given by ΔC_T, where ΔC_T is determined by subtracting the average endogenous gene C_T value from the average target gene C_T value. [C_T target gene - C_T average (endogenous gene)]. The calculation of ΔΔC_T involves subtraction of ΔC_T value for the controls from the ΔC_T value for the cases [ΔC_T target gene_(case) - ΔC_T target gene_(control)]. 2^-ΔΔCt is the relative expression of the target gene in cases compared to controls.

For comparisons (between survivors and non-survivors with invasive pneumococcal disease, and healthy afebrile controls), the Mann Whitney test was used to compare two groups and Kruskal Wallis to compare three groups. Values of p < 0.005 were considered statistically significant. SPSS statistical software version 14 (SPSS, Chicago, IL) was used for all statistical analyses.