Selected QC metrics for LL, PAX GC, and TEM GC arrays.

<p>Scale factor (SF), rawQ, background (BG), percentage present (%P), total signal, actin signal, as well as actin 3′ to 5′ ratio are all listed for LL, PAX GC and TEM GC arrays used for this study. Three arrays in total did not pass quality control and were not analysed: one array was extracted by the LL method, one array by TEM GC and one from TEM.</p

Quality control: globin levels in arrays.

<p>Data were GCRMA normalised and levels of 3 representative alpha globin probe sets (A, B and C) and one beta probe set (D) were averaged for each condition. Expression level box plots indicate that in both the cases of alpha and beta globin, TEM and PAX exhibit highest levels of globin mRNA while TEM GC and PAX GC were the lowest, LL levels were generally lower than PAX and TEM, but higher than PAX GC and TEM GC.</p

Functional annotation clustering carried out in DAVID for differentially regulated genes from a cohort of ALS patients and controls, using RNA extracted from blood with LL and TEM GC undergoing PUMA GEP analysis.

<p>In the case of LL, out of 383 clusters, the top 15 are ranked in order of enrichment score (upper section). In the case of TEM GC, out of 439 clusters, the top 15 are ranked in order of enrichment score (lower section). Common clusters between LL and TEM GC are labelled in italic and underlined.</p

PCR primer sequences designed for genes validated in this study.

<p>Samples of cDNA were diluted 1∶10, and 2.5 ul were used per PCR reaction for all genes except EHBP1, where 4 µl was used with 5x Brilliant III Ultra Fast SYBR Green QPCR Master Mix (Agilent). In all cases primer concentrations were optimised to 300 mM (S, sense strand; AS, antisense strand)</p

Characteristics of subjects taking part in the study.

<p>Table indicating patient and control IDs used for this study, as well as blood sample extraction date, age, gender, diagnosis and whether patient was treated with Riluzole. Controls that were matched to patients are also indicated (M = male, F = female, R = treated with Riluzole, P = partner, NBLD REL = non-blood relative). Samples from underlined patients and controls were used in PCR validation.</p

Initial quality control for the 75 microarrays.

<p>Analysis of array data of all 75 samples run was carried out in Affymetrix Expression Console. PolyA (A) and Hyb (B) controls show that the preparation steps were consistent, and that in general all hybridisations were consistent, but that hybridisation of PAX samples differed from the rest. The % present call (C) values were generally consistent in LL, TEM GC and PAX GC samples, but were clearly lower with TEM and PAX gene samples. The scale factor between chips were generally low, except for PAX hybridisations (D) while the relative log expression (E) of all chips were generally similar with a few exceptions, and a slight general increase in PAX samples (FU, fluorescent units).</p

Representative Bioanalyzer electropherograms after initial RNA extractions.

<p>High quality RNA (RIN >7.0) can be extracted from all three methods. PAXGENE however requires a DNase step as traces from initial extractions show genomic DNA contamination at high molecular weights and as a “shoulder” to the 28S peak (indicated by asterisks). Good quality RNA can be detected after globin depletion with both TEM and PAXGENE. However, PAXGENE samples showed consistently lower concentration levels, as indicated by the smaller 18S and 28S peaks (FU, fluorescent units). Traces are representative and from single samples from each extraction method.</p

Selected QC metrics for PAX and TEM arrays.

<p>Scale factor (SF), rawQ, background (BG), percentage present (%P), total signal, actin signal, as well as actin 3′ to 5′ ratio are all listed for PAX and TEM arrays used in this study. Three arrays in total did not pass quality control and were not analysed: one array was extracted by the LL method, one array by TEM GC and one from TEM.</p

Gene expression analysis.

<p>PCA plotted using QLUCORE OMICS Explorer on GCRMA normalised data: each dot represents an array and takes into account the expression levels of every probe set and shows 2 main clusters (A). One tightly clustering ‘globin negative’ cluster includes the following conditions: LL, TEM GC and PAX GC, while a second more variable cluster “globin positive” includes arrays hybridised with PAX and TEM extracted RNA. Focusing on the globin negative group PUMA analysis identified differentially regulated probe sets (ALS patients vs controls) for the following (B; LL, 3047; TEM GC, 3619; PAX GC, 4511). A Venn diagram comparing these lists shows that TEM and PAX share more genes in common, than either with LL, and 142 genes in common between all 3 methods).</p

Representative Bioanalyzer electropherograms after IVT and labelling, pre- and post- fragmentation.

<p>After IVT, the aRNA profiles from each method showed distinctly different profiles. Generally, IVT resulted in a wide range of detectable products. However in the cases of TEM and PAX especially, distinct high intensity peaks were observed in all cases (black arrows). These disappeared in all cases in the globin mRNA depleted samples. After fragmentation, aRNA profiles were indistinguishable from each other (FU, fluorescent units). Traces are representative and from single samples from each extraction method.</p