Real-Time PCR

Please contact nucleicacid@osumc.edu or call (614) 688-5810.

The function of the Real Time PCR Shared Resource is to provide a centralized resource for state-of-the-art equipment, reagents, expertise, and education.

The facility uses the ABI Prism 7900HT Sequence Detection System and the Applied Biosystems 7900HT Fast Real-Time PCR System. These are high-throughput real-time PCR systems that detect and quantitate nucleic acid sequences. Both block configurations, the 96-well block and the 384-well block are available.

Key applications include gene expression quantitation (absolute and relative) and the detection of SNPs using the fluorogenic 5’ nuclease assay. Both chemistries, the TaqMan™ and SYBR Green, are supported. Today, several companies provide millions of pre-designed, easy-to-use gene expression and SNP genotyping assays for human, mouse, and rat. Applied Biosystems’ TaqMan™ MicroRNA Assays and Ambion’s mirVana™ qRT-PCR miRNA Detection Kits and Primer Sets are routinely used in our lab for the validation of expression profiling data. Northern analysis will eventually be replaced by this method because of the sensitivity and the dynamic range.


Absolute Quantification

This assay is used to quantify unknown samples by interpolating their quantity from a standard curve.
As an example, the absolute quantification might be used to correlate viral copy number with a disease state. It is of interest to the researcher to know the exact copy number of the target RNA in a given biological sample in order to monitor the progress of the disease.

Requirements:

  • Standard - any stock DNA or RNA of a known concentration containing the appropriate target in a serial dilution that encompasses the unknown quantities.
  • No Template Control (NTC) - a negative control that lacks template.
  • Unknown sample – sample’s quantity value is unknown but is within the range of given standards.

Relative Quantification

This assay is used to analyze changes in gene expression in a given sample relative to another sample, such as an untreated control sample. As an example, relative quantification might be used to measure alterations in gene expression in response to a chemical (drug). Quantification results for the target gene are normalized to a control gene in order to account for sample-to-sample variability; as such, relative quantification does not require equivalent amounts of input cDNA for each sample. In cases where the target and control genes have similar amplification efficiencies, relative levels of target gene expression in samples can be determined without the use of standard curves.

Requirements:

  • Target sequence - sequence that is being studied.
  • Calibrator - a sample used as a basis for comparative results (for example, untreated control).
  • Endogenous control - a gene that is present at a consistent expression level in all experimental samples; accounts for sample-to-sample variation with respect to input cDNA.

Each sample type (for example, each tissue in a study comparing multiple tissues) is amplified by the endogenous control, after which results for the target gene are normalized to the endogenous control results.

Calculation methods for relative quantification:

Relative Standard Curve Method
Comparative Ct Method


Determining which method to use

Both methods yield expression data in terms of sample-to-sample fold changes. However, in order to use the comparative Ct (threshold cycle) method, a validation experiment must be run to show that the efficiencies of the target and endogenous control amplifications are approximately equal.

The advantage of using the comparative Ct method is that the need for a standard curve is eliminated. This increases throughput because wells no longer need to be used for the standard curve samples. It also eliminates the adverse effect of any dilution errors made in creating the standard curve samples.


Multiplexing

To amplify the target and endogenous control in the same tube, limiting primer concentrations must be identified and shown not to affect Ct values. By running the two reactions in the same tube, throughput is increased and the effects of pipetting errors are reduced.

Note: Running the target and endogenous control amplifications in separate tubes using the standard curve method of analysis requires the least amount of optimization and validation.


End-Point 7900HT Applications

Allelic Discrimination (SNP Detection):

This assay indicates the genotype of samples. Perform high-throughput genotyping of bi-allelic single nucleotide polymorphisms or small insertions/deletions in genomic DNA.

Plus/Minus Scoring:

Indicates the presence or absence of a specific target sequence in a sample.


Procedure for purchasing reagents and plates from the Core:

Contact Core Personnel by email or phone to inquire about pricing.
Bring a completed 100W form with an authorized signature and current account number.


Guidelines for running samples on the ABI Prism 7900HT sequence detection system in BRT236:

  1. Users need to schedule a run online in advance because of overbooking. Pick a time from the available time slots. A run using standard real-time PCR conditions lasts 2 hrs. Please note that if your run time is more than 2.5 hrs you can only use the 4:30 pm time slot to avoid delays for the next user.

  2. Users should start their run on time and run samples within the scheduled time.
    Cancel at least 24h prior to your scheduled run to avoid being billed.
    Prepare the plate just prior to your run (if protected from light, reactions are good for a couple of hours).

  3. Analyzed data are usually available the same day except for the last run which finishes after hours and will be analyzed the following morning.
    Centrifuge the plate in the tabletop centrifuge for 2-3 min at 1000 rpm to eliminate air bubbles. Make sure to balance the centrifuge. Use only flat optical caps. DO NOT WRITE ON THE PLATE!