- What does it mean to have a sample in the digital range?
The digital range is the template dilution range in which your results are a mix of both positive and negative wells. It is important for your sample to be within the digital range for accurate quantitation. The most accurate region occurs when you have on average one copy per reaction, which equates to ~37% negative reactions. Note: if you are analyzing two assays on the same sample, both need to be in the digital range.
- How do I know if my sample is in digital range?
In a digital PCR experiment performed on an QuantStudio™ 3D Digital PCR System, gDNA samples are diluted down to a limiting quantity, such that most individual PCR reactions contain either zero or one target molecule. The procedure for determining the optimal dilution for a sample differs depending on whether or not the target copy number per genome is known.If the target copy number per genome of your samples is known, dilute the samples so that, when loaded on a QuantStudio™ 3D Digital PCR 20K Chip, each through-hole reaction will contain approximately 0.6 to 1.6 copies of the target sequence. For example, assuming 3.3 pg/copy of a given gene are present per genome and a 865-pL reaction well volume, the stock gDNA in a given sample would be diluted down to 600 copies/μL or 1.98 ng/μL in the final reaction to give 0.6 copies per reaction well.
How to determine the target copy number per genome:
To help determine copy number per genome, collect the following information:
1. If the source or species of the gDNA is known but the genome size of the organism of interest is unknown, refer to http://www.cbs.dtu.dk/databases/DOGS/index.html to determine the size of the genome in question.
2. Once the size of the genome is known, determine the mass of the genome using the following formula:
m = ( n ) ( 1.096 × 10-21 g/bp )
where m is the genome mass in grams, and n is the genome size in base pairs.
The following example calculates the mass of the human genome using the Celera Genomics estimate of 3.0 × 109 bp (haploid):
m = (3.0 × 109 bp) (1.096 × 10-21 g/bp)
m = 3.3 × 10-12 g or 3.3 pg
The example is relevant to any gene that is present at the “normal” rate of two copies per diploid genome, such as RNase P, and provides a basis to perform a digital screening experiment to determine the optimal digital range.
If you have done qPCR and your sample has a cT value between 21 and 25, then your sample is probably in the digital range.
- If my sample isn’t in the digital range or I haven’t done qPCR, what should I do?
If the target copy number per genome is unknown, say for a locus of unknown copies per genome or RNA of unknown expression level, we recommend that you determine the optimal dilution by loading and imaging a dilution series of each sample at the expected digital range. By assaying three to four data points above and below the expected digital range, you ensure that one of the data points is within the optimal digital range. Should real-time data be available for the assay and sample being used, this can guide the starting and end point of the dilution series.
- How many chips should I run on each sample?
Life Technologies standard protocol is to run 2 chips/sample. This helps average out differences due to pipetting. Depending on your application, you may decide to run only one chip/sample. Very low copy number experiments gain more power by spreading the sample across multiple chip.
- What is the difference between absolute and relative quantification?
Absolute quantification simply counts the number of molecules present. Relative quantification uses a second assay which measures the number of copies of a gene of known quantity (i.e. TERT or RNAse P only have one copy in the genome), to calculate the amount number of copies of the assay of interest when compared to the number of copies of the known assay.
- Which dyes can I use with the QuantStudio 3D?
The QuantStudio® 3D Digital PCR instrument is capable of detecting dyes that emit at 520 ± 10 nm (e.g., FAM™, intercalating dyes such as SYBR® dye), 558 ± 7 nm (e.g., VIC®), and 630 ± 30 nm (e.g., ROX™ dye, which is used as an internal reference dye). Because digital PCR is an endpoint solution and does not measure signal in real time, it is important to have confidence in endpoint readouts, and thus, our protocols utilize TaqMan® assays to ensure amplicon specificity. Intercalating dyes can be used; however, it is important to have a method of verification. We recommend and support TaqMan® probe–based methods, which are much more specific. For laboratories that are budget-conscious or have already established SYBR® primers, a working R&D protocol is available.
- How is the assay used in dPCR different from the assay used in qPCR?
The assay used in dPCR is the same as the assay used in qPCR. Taqman assays are recommended because of their specificity, but SYBR green assays may also be used, if the customer is certain there are no primer-dimer interactions which will conceal all of the positive results.