Presented at:
2005 Meeting of the Federation of Clinical Immunology Societies, Boston, MA, May 12-16, 2005.
Comparison of RNA Preparation Methods and Their Effect on Cytokine/Chemokine Gene Expression
Asare AL, Kolchinsky SA, Wood P, Seyfert-Margolis VL.
Immune Tolerance Network, Bethesda, MD; University of Pittsburgh, Pittsburgh, PA
Objective : Commercially available blood collection tubes for gene expression studies lyse red blood cells and stabilize RNA to ensure detected transcript profiles reflect the physiological state of the subject. We assessed two such systems for their ability to detect and quantify differential expression of cytokine and chemokine genes.
Method: Peripheral blood samples from 8 healthy volunteers were collected in both Tempus Tube™ blood collection tubes (Applied Biosystems (ABI), Foster City , CA ) and PAXgene ™Blood RNA Tubes ( PreAnalytiX , Valencia , CA). All samples were isolated according to the manufacturers' instructions. RNA purity and yield were assessed prior to analysis. Samples were preprocessed as follows: 1) Blood was drawn directly into Tempus™ and PAXgene™ tubes or 2) Samples were drawn in LI Heparin tubes, stimulated with PHA, and then transferred into the two RNA collection tube types. We analyzed both: 1) Transcript detection at single time-points and 2) Differential expression between baseline samples and samples stimulated with 25 μg/ml PHA after 3 hrs. Affymetrix HG-U133 2.0 GeneChip® with 47K transcripts and quantitative RT-PCR for 184 cytokine and chemokine transcripts (probe primers from ABI) were run.
Results: Initial analysis of 2 out of 8 participants generated a GeneChip® analysis set of 1,245 reliably detected transcripts. Clustering using standard correlation generated the following differential expression profiles: Set A (370 transcripts) showing up regulation using both tubes; Set B (336 transcripts) showing down regulation using both tubes; Set C (180 transcripts) showing no change, but having elevated baseline values in Tempus™ but not PAXgene™; Set D (249 transcripts) showing no change, but having elevated baseline values in PAXgene™ but not in Tempus™. Transcripts in Set C and D were not cytokine/chemokine related. Transcripts in Set A with greater than 10 log scale expression include chemokine (c motif) ligand 1, class 1 MHC-restricted T cell associated molecule, chemokine (C-X-C motif). Quantifying differential expression by RT-PCR showed high similarity between the tubes for all cytokine/chemokine transcripts. Some allergy relevant cytokines, such as IL-5, were not detectable at high levels in the GeneChip® assay, but were detected at high levels using RT-PCR.
Conclusion: Different kinetics for each tube during the lysing process may account for detection level differences at single time-points for non-cytokine/chemokine related transcripts found using GeneChips®. These time-point differences could not be confirmed using RT-PCR since cytokine/chemokine RT-PCR probes were used. This demonstrates that the selection of a particular tube for RNA studies may depend heavily upon the transcripts of interest. For cytokine/chemokine transcripts, both tube types provide essentially equivalent expression profiles.