Fig 1.
Sample collection, processing and RNA extraction: Whole blood was collected in triplicate followed by peripheral blood mononuclear cells (PBMCs) separation in a subset of samples.
The following sample collection tubes were used for the study: RNAgard®, PAXgene® RNA, EDTA, ACD-A, and CPT tubes. The PBMC separation was done using standard procedures for CPT tubes, and magnetic bead, LeukoLOCK™ and LSM methods. The samples were then stored at either 4°C (Cold) or -80°C (Frozen) and shipped overnight (o/n) for follow-up RNA extraction. Next, they were treated with one or more of several different RNA extraction procedures: Biomaxi Precip Buffer/ PAXgene® Blood miRNA, PAXgene® Blood miRNA, TRIzol® LS, ACK Lysing Buffer/ Qiagen miRNeasy, and TRIzol® Reagent manufacturer’s protocol.
Table 1.
General statistics for RNA concentration (in micrograms) and RNA integrity number (RIN) data per mL of blood.
The sample collection tube followed by RNA extraction procedure is listed in the rows. The PBMC separation procedure is also referred as PBMC prep. CV refers to coefficient of variation and SD refers to standard deviation and μg refers to microgram.
Table 2.
Overview of correlation ratio, distance ratio, and RIN medians.
Red denotes more favorable values (high RIN; low distance ratio; high correlation ratio) while green denotes less favorable values. The sample type refers to whole blood and PBMCs and method explains the RNA isolation procedure and/or PBMCs separation procedure.
Fig 2.
Microarray signal intensity distribution before and after normalization.
Raw (a) and normalized (b) microarray probe M values (log2 fold differences) obtained for five different individuals using all different blood collection systems. Colors represent individual donors.
Fig 3.
Two dimensional Sammon projection using the top 50% of most variable transcripts (25370) for whole blood (red) and PBMC (black). There is a clear distinction in clusters for PBMCs and whole blood sample processing as marked by a black tilted line. All whole blood samples are shown on left side and PBMC samples are on the right side of the plot. The (F) and (C) denotes the frozen and cold conditions for the sample collection and storage. The description of all the legends is given here. PBMCs samples: ACD_Magnetic- blood collected in ACD-A tubes and separated using magnetic method followed by extraction using TRIzol® method. CPT—blood collected in CPT tubes and PBMCs separation using manufacturer recommendations followed by extraction using TRIzol® method. EDTA_LSM- blood collected in EDTA tube and PBMC separation using LSM followed by extraction using TRIzol® method. EDTA_Leukolock- blood collected in EDTA tube, PBMCs separated using LeukoLOCK™ method followed by extraction using TRIzol® method. Whole blood samples: ACD- blood collected in ACD-A tube followed by RNA extraction using TRIzol® LS method. ACD_RBC lysis- blood collected in ACD-A tube followed by RBC lysis using ACK buffer and RNA extraction using miRNAeasy extraction kit. EDTA_RBC lysis -blood collected in EDTA tube followed by RBC lysis using ACK buffer and RNA extraction using miRNAeasy extraction kit. EDTA- blood collected in EDTA tube followed by RNA extraction using TRIzol® LS method. PAXgene- blood collected in PAXgene® tube followed by RNA extraction using PAXgene® blood miRNA kit. RNAgard—blood collected in RNAgard® tube followed by RNA extraction using Biomaxi™ Precip Buffer and PAXgene® Blood miRNA kit.
Fig 4.
Sammon mapping using Manhattan distance of all probes for PBMCs sample separation methods where non-frozen LeukoLOCK™ separated (red) and cryopreserved samples (black) are shown as two distinct groups. The legends are described Fig 3. The CPT tubes and LSM separated PBMCs are clustered together whereas magnetic separation and LeukoLOCK™ separated PBMCs are clustered as distinct clusters with all clusters marked using a dotted line. The (F) and (C) denotes the frozen and cold conditions of the sample storage before RNA extraction.
Fig 5.
Sammon mapping using Manhattan distance of significant probes (p<0.05) for whole blood sample preparation methods where non-preserved/cold conditions (red) and frozen/cryopreserved samples (black) are shown as two distinct groups and are shown separated by a black line. The legends are described in Fig 3.
Fig 6.
(A) Venn diagram showing the common number of DEGs where comparisons of PBMCs and whole blood are affected by cryopreservation (frozen) vs. cold conditions are done. (B) Venn diagram showing the common DEGs from top 1000 mapped IDs using IPA where comparisons of PBMCs and whole blood are affected by cryopreservation (frozen) vs. cold conditions.
Table 3.
The functions associated with hematological system development: Top 1000 FDR corrected DEGS from whole blood when frozen vs. cold conditions are compared using BH p-value (Benjamin-Hochberg corrected p-value) settings in IPA.
Fig 7.
Hierarchical heatmap with diseases and bio-function category and top 5 high level functions are displayed and labeled inside the highlighted green border.
The visualization is a TreeMap (hierarchical heatmap) where the major boxes represent a family (or category) of related functions. Each individual colored rectangle is a particular biological function or disease and the color indicates associated log of the calculated BH corrected p-value: lower p value (purple), or higher p value (white). Darker colors indicate lower p values. In this default view, larger squares indicate more significant overlap between the genes perturbed in the dataset and the function or disease. The image has been cropped for better readability. Here data from whole blood when frozen vs. cold conditions are compared using top 1000 FDR corrected DEGs was used.
Table 4.
Biofunctions and diseases in the order of p-value affected in whole blood when frozen vs. cold conditions are compared using top 1000 FDR corrected DEGs.
BH p-value refers to the Benjamini-Hochberg corrected p-value in IPA.