Extensive detail on the application of the real-time quantitative polymerase chain reaction (QPCR) for the analysis of gene expression is provided in this unit. The protocols are designed for high-throughput, 384-well-format instruments, such as the Applied Biosystems 7900HT, but may be modified to suit any real-time PCR instrument. QPCR primer and probe design and validation are discussed, and three relative quantitation methods are described: the standard curve method, the efficiency-corrected DeltaCt method, and the comparative cycle time, or DeltaDeltaCt method. In addition, a method is provided for absolute quantification of RNA in unknown samples. RNA standards are subjected to RT-PCR in the same manner as the experimental samples, thus accounting for the reaction efficiencies of both procedures. This protocol describes the production and quantitation of synthetic RNA molecules for real-time and non-real-time RT-PCR applications.

Summary After infecting peripheral sites, herpes simplex virus (HSV) invades the nervous system and initiates latent infection in sensory neurons. Establishment and maintenance of HSV latency requires host survival, and entails repression of productive cycle (“lytic”) viral gene expression. We find that a neuron-specific microRNA, miR-138, represses expression of ICP0, a viral transactivator of lytic gene expression. A mutant HSV-1 (M138) with disrupted miR-138 target sites in ICP0 mRNA exhibits enhanced expression of ICP0 and other lytic proteins in infected neuronal cells in culture. Following corneal inoculation, M138-infected mice have higher levels of ICP0 and lytic transcripts in trigeminal ganglia during establishment of latency, and exhibit increased mortality and encephalitis symptoms. After full establishment of latency, the fraction of trigeminal ganglia harboring detectable lytic transcripts is greater in M138-infected mice. Thus, miR-138 is a neuronal factor that represses HSV-1 lytic gene expression, promoting host survival and viral latency.

This unit describes the use of real-time quantitative PCR (QPCR) for high-throughput analysis of RNA expression. The topics covered include: design and validation of QPCR primers and probes for both SYBR Green-and TaqMan-based assays (see Support Protocol); the standard curve method (see Basic Protocol 1); an efficiency-corrected Ct (cycle time, also called cycle threshold or crossing point) method (see Basic Protocol 2); and the comparative cycle time, or Ct method (see Alternate Protocol). While the unit describes the use of the Applied Biosystems 7900HT (high-throughput, 384-well) instrument, the protocols may be utilized for any real-time PCR instrument. The highthroughput design allows analysis of the levels of transcript from a number of genes of interest (GOIs) at one time by using the appropriate primer set for each gene. (Within this unit, the term GOI will refer to the actual gene of interest as well as its RNA product or cDNA copy.)Because of the simplicity of the mathematical application, the standard curve method (Basic Protocol 1) is the most basic and straightforward QPCR assay described in the unit. In this method, standard curves are constructed for all of the GOIs from which RNA expression is being measured, and linear regression analysis is applied to interpolate arbitrary unknown sample values. The standard curve assay may be performed even if the PCR amplification efficiencies of the primer sets (as determined by the template dilution assay in the Support Protocol) are not equal, since correction for unequal efficiencies is intrinsic to the linear regression formula. One drawback of the standard curve method is that standard curves must be run for each of the primer sets on an assay plate. This results in less space on the plate for the unknown samples, and requires the use of additional reagents. This use of resources is particularly excessive when the PCR amplification efficiencies of the primer sets have been determined to be 100% and relative fold-change is the preferred outcome of the measurements. In such cases, the Ct method (see Alternate Protocol) should be employed instead. Another limitation is that unless the levels of all of the GOIs in the cDNAs used to construct the standard curves are known, the relative concentration of one GOI cannot be compared to that of another GOI. If comparison between the levels of different GOIs (without the knowledge of the relative level of the transcripts in the standards) is desired, the efficiency-corrected Ct method (see Basic Protocol 2) should be applied.The efficiency-corrected Ct method builds upon the standard curve method by incorporating PCR efficiency (E) into the quantity calculations. The standard curve slopes are used to calculate PCR efficiency according to the relationship E = 10 (-1/slope) . The efficiency has a maximum value of 2 for perfect doubling of the PCR template (see Basic Protocol 2 for an in-depth explanation of E). Note that in this method the standard curve is used only to determine slope and not to interpolate the RNA...

Genetic studies in chickens and receptor interference experiments have indicated that avian leukosis virus (ALV)-E may utilize a cellular receptor related to the receptor for ALV-B and ALV-D. Recently, we cloned CAR1, a tumor necrosis factor receptor (TNFR)-related protein, that serves as a cellular receptor for ALV-B and ALV-D. To determine whether the cellular receptor for ALV-E is a CAR1-like protein, a cDNA library was made from turkey embryo fibroblasts (TEFs), which are susceptible to ALV-E infection, but not to infection by ALV-B and ALV-D. The cDNA library was screened with a radioactively labeled CAR1 cDNA probe, and clones that hybridized with the probe were isolated. A 2.3-kb cDNA clone was identified that conferred susceptibility to ALV-E infection, but not to ALV-B infection, when expressed in transfected human 293 cells. The functional cDNA clone is predicted to encode a 368 amino acid protein with significant amino acid similarity to CAR1. Like CAR1, the TEF protein is predicted to have two extracellular TNFRlike cysteine-rich domains and a putative death domain similar to those of TNFR I and Fas. Flow cytometric analysis and immunoprecipitation experiments demonstrated specific binding between the TEF CAR1-related protein and an immunoadhesin composed of the surface (SU) envelope protein of subgroup E (RAV-0) virus fused to the constant region of a rabbit immunoglobulin. These two activities of the TEF CAR1-related protein, specific binding to ALV-E SU and permitting entry only of ALV-E, have unambiguously identified this protein as a cellular receptor specific for subgroup E ALV.Retroviral infection is initiated through interactions between the viral envelope protein (Env) and specific receptors present on the surface of the host cell. The viral surface (SU) Env protein directly binds to the viral receptor, and subsequently conformational changes in Env that expose fusion peptide regions of the transmembrane (TM) Env protein are thought to drive fusion of the viral and cellular membranes (1, 2). We are using avian leukosis virus (ALV) receptor interactions as a model system to understand how retroviruses enter their host cells. There are six well characterized chicken subgroups of ALV (A-E and J) which are defined on the basis of host range, antibody neutralization, and receptor interference studies (1).Subgroups B, D, and E of ALV are predicted to utilize the same or related receptors in susceptible chicken cells. One line of evidence that supports this hypothesis comes from receptor interference studies. Cells preinfected with either ALV-B or ALV-D are resistant to superinfection by subgroup B, D, and E viruses, presumably because of newly synthesized viral Env binding to the receptor, thus preventing subsequent rounds of viral entry (1). However, cells preinfected with ALV-E are resistant only to superinfection by subgroup E viruses. The reason for the nonreciprocal receptor interference pattern exhibited by these viruses remains to be determined.Further evidence that these viruses us...

Several herpes simplex virus 1 microRNAs are encoded within or near the latency associated transcript (LAT) locus, and are expressed abundantly during latency. Some of these microRNAs can repress the expression of important viral proteins and are hypothesized to play important roles in establishing and/or maintaining latent infections. We found that in lytically infected cells and in acutely infected mouse ganglia, expression of LAT-encoded microRNAs was weak and unaffected by a deletion that includes the LAT promoter. In mouse ganglia latently infected with wild type virus, the microRNAs accumulated to high levels, but deletions of the LAT promoter markedly reduced expression of LAT-encoded microRNAs and also miR-H6, which is encoded upstream of LAT and can repress expression of ICP4. Because these LAT deletion mutants establish and maintain latent infections, these microRNAs are not essential for latency, at least in mouse trigeminal ganglia, but may help promote it.