FAQ

All of our assays are thoroughly tested on complex biological samples (i.e cell or tissue cDNA samples) and specificity is determined in real qPCR reactions. We run a melt curve, and also an agarose gel to check for a single band of the correct size. In addition, for some of our assays (e.g. all the human ones and many of the mouse ones) the PCR products have also been sequenced. The BLAST search in the primer report shows potentially unintended templates. The primer report includes any templates that show enough similarity in the primer region to potentially amplify, but this does not mean that the assay is not specific under experimental conditions. For example, some potentially unintended templates identified by BLAST may be too long to be successfully amplified under the qPCR assay conditions, or may have a 3′ base mismatch. The primer-BLAST report is a prediction based on bioinformatic algorithms, our standards are tested in real biological samples.

The copy number (number of molecules of amplicon) is calculated from the absorbance at 260 nm using the extinction coefficient for each specific product (amplicon). Extinction coefficients are both sequence-dependent and product length-dependent, and this therefore is the most accurate method for determination of copy number. The maximum calculated error in copy number is 5%. We have validated our method of quantifying the copy number by digital droplet PCR, which produced an empirically-determined mean error of 5.1%, similar to the predicted error. Furthermore we observe loss of linearity upon dilution of the standard to below 10 copies per reaction; the Cq becomes highly variability and we observe unsuccessful amplification in some samples (due to the random nature of sampling very dilute solutions).

We recommend either nuclease-free water or yeast tRNA. If water is used, we strongly recommend storing aliquots in ‘no-stick’ tubes to prevent adhesion to the tube walls and therefore loss of the some of the standard, and particularly of the diluted working solutions, upon storage. tRNA acts as a carrier to prevent DNA loss from sticking to the tube walls. There is also some evidence that tRNA can help to reduce the formation of primer dimers during PCR (Sturzenbaum, 1999). EDTA-containing buffers are not recommended as they may interfere with the performance of the DNA polymerase by chelating magnesium ions.

Reference: Sturzenbaum SR (1999) Transfer RNA reduces the formation of primer artifacts during quantitative PCR. Biotechniques 27:50-52.

Our standards are shipped dry and may be stored at room temperature until reconstituted. Once reconstituted, we recommend making conveniently-sized aliquots and storing at -20°C. We recommend that the standards be reconstituted to make a stock solution of 1×10⁸copies μL^-1, however, if they are to be distributed amongst multiple users, an initial reconstitution to 1×10⁷copies μL^-1 may be more practical. Full instructions are provided with each assay. Standards may be thawed several times without loss of performance and are stable for one year once reconstituted as long as the recommended storage conditions are followed. If not reconstituted immediately, we guarantee the performance of the standard for up to 18 months from the purchase date. Temporary storage at 4°C is acceptable, for example, if the standard is to be used several times in one day.

All our assays have been designed and tested at an annealing temperature of 57°C routinely or 60°C upon request. Some assays have been tested at both temperatures. We do not recommend annealing temperatures lower than 57°C. We have tested standards in both a three-step cycling protocol, and a two-step protocol where the annealing and extension steps are combined into one step. Other parameters such as time, and denaturation and extension temperatures are a property of the PCR mix, specifically the DNA polymerase and buffer, and so the manufacturer’s recommendations should be followed. For some qPCR machines there is also a lower restriction on the minimum annealing/extension period time to allow the fluorescence data to be collected.

The cycle at which the SYBR green fluorescence of the PCR products increases over that of the background, commonly referred to as the Cq, is affected by many factors, one of which is the sensitivity of the detector on the qPCR cycler. The instrument on which the standards were tested is particularly sensitive compared with some other qPCR cyclers on which our standards have been run. We have observed a Cq of up to 3 cycles earlier on our machine. We have not found this difference between machines to adversely affect performance of the standards.

Our standards have been used with a number of different PCR mixes and we do not recommend any specific manufacturer. You may continue to use your preferred mix, or the formulation recommended for your real-time cycler. We have recently tested over 90 of our assays using Brilliant III from Agilent with a 1 second combined annealing and extension step and observed excellent sensitivity, linearity and efficiency, even under these extremely short cycling conditions.

All our assays have been verified to be linear to 10 copies. Any complimentary primer aliquots provided with the assay are template-free (no amplification in the NTC). Non-linearity means that you have contaminated your water, primers or qPCR mix with template and there are therefore more copies present than there should be. This mostly likely originates from your cDNA samples or the standards. It is recommended to always spin down tubes of standards or cDNA prior to opening them and to never open them in the presence of open vials or tubes of water, primers or qPCR mix. Prepare the master mix first and cap the tube before carefully opening the standards or cDNA sample tubes. The standards contain very high copy numbers of amplicon and even a single copy will produce a positive amplification curve in a reaction. Should you observe contamination, re-dilute the standards using fresh water or tRNA starting from the 10⁷dilution. Use fresh working solutions of primers and a fresh aliquot of qPCR mix.

In our experience this is often, but not always, the case. The melt peak depends more on amplicon sequence (specifically, GC content) than length, whereas sequence does not affect the migration of PCR products on a gel as this is length-dependent. A gel can therefore sometimes resolve more than one product in a sample that appears as a single peak on a melt curve. All of our assays are checked to ensure a unique product is generated by running the PCR products on a gel as well as checking the melt curves.

Even if equal amounts of similar quality RNA are reverse transcribed, the reverse transcription step itself can be very variable from sample to sample within the same experiment and even between reverse transcription replicates of the same RNA sample. Moreover, reverse transcription is typically less than 50% efficient. This is one reason that data normalisation using a panel of reference genes is so important as it corrects for variability in the sample that is due to factors other than the experimental conditions. The purpose of qPCR standards is rather for more pragmatic reasons – see ‘What’s the advantage of using qPCR standards over relative quantification?’. qPCR standards for a range of commonly used reference genes for various species are available from qStandard.

An intron-spanning assay is one where at least one of the primers is located in two different exons across an exon-intron junction. This primer will therefore only anneal perfectly to cDNA derived from spliced mRNA (mature transcript) and not to genomic DNA, or to cDNA derived from unspliced mRNA (immature transcript), because in the latter two the intron will be present and will disrupt complementary base-pairing for part of the primer sequence, effectively reducing its annealing temperature. An intron-flanking assay is one in which the two primers are located in different exons but not across an exon-intron junction. In this case any product arising from cDNA derived from spliced mRNA (mature transcript) will give the correct product; any product amplified from genomic DNA or cDNA derived from unspliced mRNA (immature transcript) will be longer than expected (if the intron is short enough to be amplified under the cycling conditions). Extremely long introns (thousands of bases) are unlikely to amplify, but many modern engineered DNA polymerases are fast and can certainly extend several hundred bases in a few seconds.

We would usually run at least five, but recognise that in some situations this may not be practicable – for example when only very low amounts of RNA are extracted from limited sources. Using normalisation software such as geNorm or Normfinder, the most stable reference genes can then be selected to normalise data. Once the most stable reference genes are established for a given set of experimental conditions, only those genes need be run in subsequent experiments.