High-throughput quantitative real-time RT-PCR assay for determining expression profiles of types I and III interferon subtypes.

Described in this report is a qRT-PCR assay for the analysis of seventeen human IFN subtypes in a 384-well plate format that incorporates highly specific locked nucleic acid (LNA) and molecular beacon (MB) probes, transcript standards, automated multichannel pipetting, and plate drying. Determining expression among the type I interferons (IFN), especially the twelve IFN-α subtypes, is limited by their shared sequence identity; likewise, the sequences of the type III IFN, especially IFN-λ2 and -λ3, are highly similar. This assay provides a reliable, reproducible, and relatively inexpensive means to analyze the expression of the seventeen interferon subtype transcripts.

Expression profiles of human interferon-alpha and interferon-lambda subtypes are ligand- and cell-dependent.

Recent genome-wide association studies suggest distinct roles for 12 human interferon-alpha (IFN-α) and 3 IFN-λ subtypes that may be elucidated by defining the expression patterns of these sets of genes. To overcome the impediment of high homology among each of the sets, we designed a quantitative real-time PCR assay that incorporates the use of molecular beacon and locked nucleic acid (LNA) probes, and in some instances, LNA oligonucleotide inhibitors. We then measured IFN subtype expression by human peripheral blood mononuclear cells and by purified monocytes, myeloid dendritic cells (mDC), plasmacytoid dendritic cells (pDC), and monocyte-derived macrophages (MDM), and -dendritic cells (MDDC) in response to poly I:C, lipopolysaccharide (LPS), imiquimod and CpG oligonucleotides.

Systematic method for determining an ideal housekeeping gene for real-time PCR analysis.

To standardize the amount of biological material between samples (e.g., number of cells or amount of tissue) for quantitative real-time reverse transcriptase PCR (qRT-PCR), the cycle of the target gene at which expression is detected (the cycle threshold, or Ct) is divided by the Ct of a gene either thought to be unaffected by experimental conditions or similarly expressed among donors.