Overview

A user can bring several gene names or accession numbers and a few micrograms of total RNA for each sample to the Facility, and usually receive initial quantitative expression data within several days. Publication-ready quantitative analysis with sufficient replicates can often be obtained within a week. In addition to relative gene expression analysis, Q-PCR analysis in the Facility can also provide gene, RNA, or viral copy number data.

Why do Q-PCR?

Real-time quantitative PCR (Q-PCR) is rapidly becoming very popular, because for many applications, it is far superior to older methods such as Northerns, RNase protection assays, or “semi-quantitative” PCR. Compared to Northerns and RNase protection assays, Q-PCR requires orders of magnitude less RNA, is faster, cheaper, and far more quantitative. In addition, no plasmids or probes are required for Q-PCR, only DNA sequence information of the genes being studied. Q-PCR is actually less work than semi-quantitative PCR (a technique viewed with increasing skepticism by reviewers), as during an unattended 2 hr run, Q-PCR measures PCR results after every cycle without gel analysis.

cDNA Synthesis

Typically, the user provides 4 µg of total RNA. This RNA can first be DNase treated to remove genomic DNA. Following purification of the RNA, oligo (dT) primed cDNA synthesis is performed using Invitrogen Superscript III. This typically yields sufficient cDNA for over 100 Q-PCR reactions, and can be scaled down if RNA is limiting.

Primer Design and Validation

SYBR Green Primers The vast majority of Q-PCR in the Facility is SYBR Green analysis, which requires two unmodified primers about 20 bp in length. The Primer Design Service requires only a gene name or accession number. A primer pair is then designed, purchased and tested in a Q-PCR reaction, using either a Universal cDNA sample or a sample supplied by the user. If users are designing their own primers, it is requested that they use the same parameters as the Facility so that they will be compatible with other assays. The QC process included in the Primer Design Service is an assessment of the C_t and disassociation curve (melting curve) of the product in a Q-PCR experiment. Such validation is usually sufficient for most analysis, but for publication-quality validation, two other services offered:

(1) Gel Analysis of the Q-PCR Product, to verify that the PCR product is of the predicted length and purity.

(2) DNA Sequencing of the Q-PCR product, where the PCR product is purified and the DNA sequence determined using one of the two Q-PCR primers. This verifies the specificity and identity of the amplified product, at a fraction of the cost of hybridization probe (TaqMan) analysis.

TaqMan Primers The Facility does not have extensive experience in the design of primers and hybridization probes for TaqMan assays, and it is suggested that design and purchase of these relatively expensive primers be outsourced. Applied Biosystems sells guaranteed TaqMan Gene Expression Assays for virtually every gene in humans, mice, rats, and flies.

SYBER GREEN ANALYSIS

Q-PCR from cDNA (2-step analysis). The Facility will help users design experiments that will yield significant data. In this analysis, cDNA is first generated from the total RNA sample. Working from a single preparation of cDNA allows for more uniform and controlled Q-PCR analysis, and is more robust than the single tube (1-step) reverse transcription-Q-PCR reaction. Two-step analysis is recommended if the user wants to assess the expression of a number of genes in each RNA sample. In typical analysis, a standard curve is generated with a control gene (usually Cph/PPIA), with control primers provided by the Facility. The amount of cDNA is normalized using the Cph signal, and the relative normalized expression of each gene in the experiment is determined. Data returned include a PowerPoint file with the graphical data (amplification, standard, and melting curves) and an excel table including the raw and normalized data.

Q-PCR from RNA (1-step analysis). In this approach, one starts with 10-200 ng RNA per sample (depending on abundance), and uses a reaction mix that contains both the reverse transcriptase and Taq polymerase. Priming of cDNA synthesis is by the 3’ PCR primer. Following a 30’ incubation for RT, Q-PCR then proceeds as normal. Disadvantages of this approach include the less robust reaction conditions (a compromise buffer system where both RT and Taq can function), more expensive reactions, and greater difficulty in normalization. However, there are several advantages, and if one wants to assess expression of a couple genes and parallel assays of a control gene (e.g. Cph) in many different samples, this is the way to go. In addition, because this approach uses gene-specific priming for cDNA synthesis (rather than oligo-d(T), one can analyze gene sequences far upstream from the poly(A), such as in the coding sequence of a gene with a long 3’ untranslated region. Data provided are the same as for 2-step analysis.

Q-PCR with TaqMan or other hybridization probes

The same Q-PCR services as SYBR Green (analysis from cDNA or RNA) are also offered with hybridization probes. Data are provided in the same format, except that there are no melting curves. There are many exotic Q-PCR probe systems besides TaqMan, but these mostly represent ways around the ABI patent, rather than actual improvements.

Q-PCR Multiplexing assays

Multiplexing assays for gene copy number (control and experimental) can be developed and performed in the Facility. The Facility would be happy to discuss the utility and desirability of multiplexing Q-PCR approaches for gene expression analysis. In general, multiplex Q-PCR requires substantial optimization, and is only recommended if assay samples are extremely limiting.

Absolute (copy number) Quantitation with Gene-specific Std. Curves

Most analysis of gene expression is relative analysis, based on relative expression changes of a particular gene (with a given primer pair) between different RNA samples. One can not compare Q-PCR signals between two different primer pairs and infer their relative abundance. For abundance comparison between different genes, one must generate gene-specific standard curves and determine absolute copy numbers. This is most easily achieved using standard curves with purified, quantitated PCR product. The Facility will assist users in developing such assays, and will perform the Q-PCR analysis.

Want Q-PCR, but not sure which kind?

There are two main issues where one must make a choice on what is the most appropriate Q-PCR approach. As a starting point, we usually strongly recommend SYBR green analysis (vs. TaqMan), and suggest starting with cDNA (vs. RNA) but the choice of cDNA vs. RNA depends strongly on the experiment.

1. SYBR Green vs. TaqMan. SYBR green primers are over 10-fold cheaper than the primers and probe needed for TaqMan analysis (approximately $15 vs. $200), and we have had excellent experience assaying the expression of hundreds of genes by SYBR Green Q-PCR. An objective study found similar linearity between SYBR Green and TaqMan assays. Clearly the cost difference becomes substantial where one is looking at the differential expression of many different genes (e.g. microarray follow-up or gene family assays). The TaqMan assay is potentially more specific, as the hybridization probe hybridizes to a central region of the PCR product produced by two normal primers. Taq 5’ exonuclease removal of a quencher from near the probe fluorphore then leads to fluorescence. In contrast, SYBR Green becomes highly fluorescent after it binds to any double-stranded DNA in the PCR reaction. That is why every SYBR green experiments always includes the melting curve analysis to assess PCR product purity. In addition, for genes of interest, the one-time PCR gel analysis and PCR product sequencing can be used to assure specificity. TaqMan assays can also be more sensitive, which is useful for things like detecting viral nucleic acid in the bloodstream. Finally, one can buy many pre-qualified TaqMan assays or find information for functional assays in the literature. This may save some time, as only about 90% of the SYBR green primer pairs work on the first try.

2. Q-PCR from cDNA (2-step) vs. directly from RNA (1-step). The relative merits of these approaches are discussed above. In short, if one is assaying the expression of many genes in a few RNA samples (e.g. microarray follow-up) , one should use the 2-step approach. If one is studying a few genes in many RNA samples (e.g. testing siRNAs or patient samples), then one should use the 1-step approach. Indeed, if one is analyzing the same gene in a large number of samples, then a TaqMan probe for the 1-step assays may be worth the investment. Consulting for selection of the appropriate Q-PCR assay in the Facility is available at no charge.