Cell type-specific total RNA
High-quality RNA for gene expression analysis— ready-to-use for quantitative RT-PCR or microarray analysis Reliable cell-type specific RNA— from cells separated with MACS® Technology for optimal purity and gentle processing The isolation of highly pure total RNA samples from distinct hematopoietic cell populations is laborious and costly to achieve. This is especially true for rare and immunologically relevant cell populations such as regulatory T cells (CD4+ CD25+). Each cell-type specific total RNA is a high-quality RNA derived from various hematopoietic cell populations, ready-to-use for gene expression profiling experiments, gene cloning, or as a reference for further analyses. High production standards. Using the standard method for cell isolation, MACS® Technology, specific blood cell fractions are separated from healthy donor blood. Extremely small MicroBeads and gentle MACS Column Technology enable the isolation of a subpopulation of cells with generally no effect on cell function or activity. From this specific cell fraction, total RNA is isolated. After removal of residual genomic DNA, RNA is extensively quality controlled. Ready-to-use total RNA. There is no need to collect large pools of blood and to separate the desired cell population. The high-quality RNA is ready to use—as sample or reference for gene expression profiling such as quantitative PCR or microarray analysis. Thus, differences in gene expression in various cell types are revealed. Using the total RNA as reference allows comparison of treated cell culture samples or patient samples to healthy, untreated conditions. Alternatively, the RNA can be used to clone target genes following cDNA synthesis or RT-PCR. Consistent and reliable cell type-specific total RNA • Blood donation Donors are consenting, healthy, and unmedicated adults aged 18–68 years and donations are performed according to standard legal regulations. Gender of donors is 50% female and 50% male. Donors are negative for HIV, hepatitis B and C, and syphilis. • Cell preparation Peripheral blood mononuclear cells (PBMCs) are prepared from buffy coats by density gradient centrifugation. Cell subsets are isolated from PBMCs by positive magnetic selection using the corresponding MicroBeads. Please see the brochure MACS Technology—Gold standard in cell separation for detailed information about cell separation. Cell purity exceeded 90% in all cell populations. • RNA preparation and purity Pooled, purified cells from multiple donors are lysed and total RNA is extracted using silica-membrane technology. After genomic DNA removal, RNA purity is determined by capillary electrophoresis with an Agilent Bioanalyzer. In general, the resulting RNA Integrity Number (RIN) values¹,² are 8.5 or higher. • Quantitative PCR control The Hypoxanthine-guanine phosphoribosyltransferase (HPRT) gene is a housekeeping gene that is normally expressed at 1–10 mRNA copies per cell.3 Thus, it provides an adequate reference for RNA quality. 1 ng, 10 ng, and 100 ng of the corresponding total RNA are analysed by quantitative reverse transcription (RT) PCR with intron-spanning primers to determine HPRT mRNA content. Further information Total RNA flyer [PDF; 54 KB]		Figure 1 Real-time PCR analysis of B cell (CD19+) Total RNA. A: Quantitative RT-PCR with intron-spanning primers for 88 bp Hypoxanthine-guanine-phosphoribosyltransferase (HPRT) low-copy housekeeping gene (LightCycler®, Roche) B: Melting curve data Figure 2 Real-time PCR analysis of Regulatory T cell (CD4+CD25+) Total RNA. Quantitative RT-PCR with intron-spanning primers for 88 bp Hypoxanthine-guanine-phosphoribosyltransferase (HPRT) low-copy housekeeping gene (LightCycler®, Roche). Figure 3 Electrophoretic trace of CD3+ Total RNA. 5 µg isolated and lyophilized CD3+ Total RNA was dissolved with 50 µL RNAse-free distilled water and stored at –70 °C. An aliquot of 1 µL was then analyzed using a Bioanalyzer 2100 (Agilent Technologies).

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