Increases sensitivity of gene detection in microarray profiling experiments
Integrates easily with the RiboPure-Blood Kit and MessageAmp II aRNA Amplification System for expression profiling
Total RNA from Whole Blood
The easy accessibility of blood makes it the most common tissue studied in clinical research, but blood consists of a heterogeneous population of cells, including erythrocytes, granulocytes, and other peripheral blood mononuclear cells. One obstacle for expression profiling RNA from whole blood is the relatively high content of globin messenger RNA (mRNA) from immature red blood cells. In fact, up to 70% of the mRNA mass is from globin transcripts.
Globin Transcript Removal
Ambion's GLOBINclear-Human Kit is designed to rapidly deplete alpha and beta globin mRNA from total RNA preparations derived from human whole blood. The kit employs a novel hybridization capture technology (see "Simple, Effective Globin mRNA Reduction Protocol" at right) that uses the power of biotin/streptavidin binding and the specificity of nucleic acid hybridization to effectively remove 9099.5% globin transcripts from blood total RNA (Figure 1).
Figure 1. Electropherogram of Amplified RNA from Human Whole Blood. Total RNA was isolated from whole blood using the RiboPure-Blood Kit (Ambion). Total RNA (5 g) was treated with the GLOBINclear Kit (Ambion). MessageAmp II aRNA Amplification Kit (Ambion) was used to linearly amplify 2 g total RNA or RNA enriched by the GLOBINclear Kit. The resulting aRNA was resolved on an Agilent 2100 bioanalyzer RNA LabChip assay to monitor quality and quantity. Note the complete removal of the abundant "globin spike" in the GLOBINclear-treated RNA that is present in the whole blood aRNA sample.
Successful Gene Expression Profiling
To further test the specificity of globin mRNA reduction, synthetic globin transcripts were added to HeLa cell RNA, and samples were processed or left unprocessed with the GLOBINclear Kit (Figure 2). In microarray analyses, the GLOBINclear protocol did not alter the profiles from HeLa RNA preparations. However, the GLOBINclear process was able to rescue the decreased gene detection observed with RNA samples containing high levels of exogenous globin transcripts.
Figure 2. Percent Present Calls for Samples Treated or Untreated with GLOBINclear-Human Globin mRNA Removal Kit. In a control experiment, the GLOBINclear Kit (Ambion) did not significantly alter the Percent Present Call results from RNA samples derived from a cultured cell line (HeLa cells) that does not express high levels of globin (left two bars). In contrast, the GLOBINclear Kit rescued the decreased gene detection observed when exogenous globin transcripts were spiked into the HeLa RNA samples. Total RNA was isolated from human blood samples with the RiboPure-Blood Kit (Ambion). While half of the samples were processed with the GLOBINclear Kit before RNA amplification (MessageAmp II Kit, Ambion), half of the total RNA preparations were used directly for RNA amplification. Labeled aRNA was hybridized to an Affymetrix GeneChip Human Genome Focus Array. Percent Present Calls were ~10% (~1000 genes) lower for samples that were not treated with the GLOBINclear Kit.
Globin mRNA levels can vary among donors, which can unpredictably affect RNA amplification and array results and therefore, confound interpretations that are made when comparing different samples. With the removal of globin transcripts, the enriched RNA samples become superior templates for RNA expression profiling because the presence of globin mRNA is no longer a factor in decreasing the sensitivity of gene detection on microarrays (Figure 2).
GLOBINclear-Human Globin mRNA Removal Kit
The GLOBINclear protocol is compatible with total RNA obtained from any high quality whole blood RNA isolation procedure. This kit was developed to reduce or eliminate the negative effects of globin mRNA on high density oligonucleotide expression profiling, and the resulting enriched RNA is compatible with the RiboPure-Blood Kit and the MessageAmp II aRNA Amplification System. The GLOBINclear Kit contains enough reagents to deplete globin mRNA from 20 samples of total RNA derived from human whole blo od (110 g per sample).
The GLOBINclear Protocol .
Penn Whitley, Sharmili Moturi, Robert Setterquist * Ambion, Inc.
Cat# Product Name Size 1751 MessageAmp II aRNA Amplification Kit 20 rxns 1753 Amino Allyl MessageAmp II aRNA Amplification Kit 20 rxns 1928 RiboPure-Blood Kit 40 purifications 1980 GLOBINclearHuman 20 rxns 10055 6 Tube Magnetic Stand 1 each