Expression array data generated from whole-blood total RNA samples have lower detection sensitivity compared to experiments using RNA from fractionated blood. Whole-blood RNA is easily accessible but contains high levels of globin mRNA that interfere with the accurate assessment of other genes. Globin transcripts represent up to 70% of the mRNA in whole-blood total RNA. Array analysis using total RNA with a high percentage of globin mRNA reduces signal-to-noise ratios, while increasing signal variation.
The Ambion GLOBINclear™ Whole Blood Globin Reduction Kits solve this problem with a protocol that rapidly depletes >95% of the alpha- and beta-globin mRNA from whole-blood total RNA. The resulting RNA is superior for synthesizing labeled cDNA/cRNA for array analysis and quantitative RT-PCR. Up to 50% more genes are detectable on Affymetrix® GeneChip® Arrays, and it requires no RNase H treatments, which can degrade mRNA and alter expression profiles, or expensive peptide nucleic acid (PNA) oligomers.
In a recent study, samples were collected from healthy subjects, globin mRNA was depleted using the GLOBINclear Kit, and the remaining RNA was amplified with the Ambion MessageAmp™ II aRNA Amplification Kit . Genes exhibiting consistent changes in expression on Affymetrix® HU133A 2.0 Arrays were characterized by molecular function, biological process, and cellular component. This study investigated the biological significance of genes whose expression was unmasked by globin mRNA reduction in peripheral blood. The results indicate that reducing globin led to the detection of 2652 ± 395 (28 ± 5%) additional genes per assay, while only 5 genes were masked after globin reduction.
Another study compared the effects of two globin reduction methods on gene expression measurements using RNA from whole blood and a cell line (Jurkat RNA with and without spiked globin mRNA) . The GLOBINclear Kit, which uses biotinylated globin-capture DNA oligos and streptavidin magnetic beads to deplete globin mRNA, was compared to a method that inhibits reverse transcription of globin transcripts during cDNA synthesis. The study found that although the GLOBINclear Kit requires more input RNA and time than the inhibitory method, RNA treated with the GLOBINclear Kit demonstrated better transcript detection sensitivity for cell line RNA, lower signal variation for whole-blood RNA, and more similar profiles to controls, compared to the inhibitory method.
For Research Use Only. Not for use in diagnostic procedures.
- Field LA, Jordan RM, Hadix JA, Dunn MA, Shriver CD, Ellsworth RE, and Ellsworth DL (2007) Functional identity of genes detectable in expression profiling assays following globin mRNA reduction of peripheral blood samples. Clin Biochem 40:499–502.
- Liu J, Walter E, Stenge Dr, and Thach D (2006) Effects of globin mRNA reduction methods on gene expression profiles from whole blood. J Mol Diagnostics 8(5):441–558.