Limitations of qPCR to estimate DNA quantity: An RFU method to facilitate inter-laboratory comparisons for activity level, and general applicability

The application of qPCR to estimate the quantity of DNA present is usually based upon a short amplicon (typically c.80bp) and a longer amplicon (typically c.200-300bp) where the latter is used to determine the amount of degradation present in a sample. The data are used to make decisions about a) whether there is sufficient template to amplify? b) how much of the elution volume to forward to PCR? A typical multiplex amplifies template in the region of 100-500bp. Consequently, the results from an 80bp amplicon will tend to overestimate the actual amplifiable quantity that is present in a degraded sample. To compensate, a method is presented that relates the quantity of amplifiable DNA to the average RFU of the amplified fragments. This provides greatly improved accuracy of the estimated quantity of DNA present, which may differ by more than an order of magnitude compared to qPCR. The relative DNA quantities can be apportioned per contributor once mixture proportions are ascertained with probabilistic genotyping software (EuroForMix). The motivation for this work was to provide an improved method to generate data to prepare distributions that are used to inform activity level propositions. However, other applications will benefit, particularly those where extraction and quantification are bypassed: For example direct PCR and Rapid DNA technology. The overall aim of this work was to provide a method of quantification that is standardised and can be used to compare results between different laboratories that use different multiplexes. A software solution “ShinyRFU” is provided to aid calculations.


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The notion of using average peak height (RF U ) of a DNA profile as a 33 proxy measurement for DNA quantity was introduced by Tvedebrink et al whereas for low template, it may not be possible to achieve the target 116 amount, hence the profile will be lower quality and/or partial.   The RF U response is dependent upon a number of factors other than the 132 quantity of DNA analysed in the PCR reaction. Different instruments will 133 have different sensitivities. Haas et al [13] showed that there was a big dif-134 ference between peak heights generated by Genetic Analysers 3500 and 3730 135 vs. 3130xl. This necessitated that RFUs from the latter were multiplied by a factor of three to standardise the output. Additional dependencies include: 137 the multiplex used; CE injection time, volume, and voltage settings, which 138 must be maintained as constant for the series of experiments or casework us-139 ing the same conditions. For a given set-up, to be able to convert RF U into 140 DNA quantity, it is necessary to carry out calibration using known quanti-141 ties of undegraded DNA. Furthermore, since there are many variables, this 142 calibration will be laboratory-specific. A dilution series was prepared with input DNA quantities ranging be- where r is the replicate index and R is the total number of replicates and 168 replicates are based upon the same multiplex.

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The relationship between the two variables is described by a log-linear is therefore more sensitive. This increased sensitivity is attributed to an 181 additional PCR cycle.
182 Figure 1: Comparison of log 10 (ng/µl) DNA quantification value versus log 10 (average RFU) for controls processed using two different multiplexes -Fusion 6C and ESX17 We define an ordinary linear regression model with log 10 RF U i as response 183 and DNA quantity log 10 Q i as explanatory variable, where i is an observation: where noise i is independent identically distributed as normal with expecta-185 tion zero and constant variance.

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The prediction for the response variable for a input DNA quantity log 10 Q, 187 is then given as the expectation: 188 log 10 RF U = log 10 a + b × log 10 Q where a = intercept and b = regression coefficient, which must be estimated.

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Since the control samples comprise pristine DNA ( fig. 1 All that is needed to carry out the conversion to DNA quantity is a multiplex 196 specific intercept regression coefficient (a) ( Table 1).    Data-set B also followed the same gradient, but the intercept a value was 235 reduced. This is because the DNA was more degraded compared to data-set The aim is to provide an optimal amount of DNA into the pre-PCR set-244 up. If too much DNA is loaded onto a CE instrument, then the signal will be 245 saturated and the profile will be poor quality. This optimum level may vary 246 between laboratories, but is typically 1ng, defined as the total amount of 247 DNA that is provided to the pre-PCR set-up; measured by a quantification 248 method, and provided in a constant volume (T el max ). However, for our subsequent calculations, we must refer to the concentration of DNA that is 250 available in the orginal (undiluted) extract. Furthermore, it is necessary to 251 adjust the calculated RF U value so that it accurately reflects the expectation 252 from the undiluted stock extract. To achieve this, it is a dilution factor (dl) 253 is calculated that is multiplied by the RF U value. for the PCR reaction, T el is taken from elution volume and added to T dl of 259 dilution buffer or water. The dilution factor is calculated: added to the PCR set-up volume of T dl . The dilution factor is calculated:  The calculated RF U is multiplied by the dilution factor to give the ad- give: procedures, e.g., multiplexes, it is necessary to carry out normalisation. To By rearrangement, log 10 RF U 2 is normalised : In this example, both b 1 and b 2 =1, hence the equation simplifies to: 301 log 10 RF U 1 = (log 10 RF U 2 − log 10 a 2 ) + log 10 a 1 (13) or without logs: may be extracted from swabs in order to provide the elution volume (E V ).

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The same calculation is carried out to normalise the DNA concentration: Because E V = 100µl is used as the standard volume, everything is normalised     In this example, a stain is extracted into a total elution volume E V = 556 100µl. A portion is taken for quantification using qPCR and the concentra-557 tion is recorded as Q i = 0.01ng/µl or a total of Q tot = 0.01 × 100 = 0.5ng.

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This value serves as a guide to optimise the amount of DNA forwarded to 559 the PCR set-up. With this example, 1ng total is optimal.

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In the PCR set-up, the total PCR volume T V = 50ul where T pcr = 35ul 561 consists of PCR mastermix and primers. The remainder of T dl + T el = 15ul 562 consists of water and DNA template respectively. Since the qPCR estimate 563 is 0.01ng/µl, it is only possible to take a maximum of 15µl x 0.01ng/µl = 564 0.15ng in total i.e. no water is added to the PCR set up volume with this 565 example.

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It is assumed that all PCR amplifications are carried out using equal 567 total PCR volume T V = T pcr + T el + T dl . Further more, the CE injection 568 parameters are assumed to be constant. Taking the same variables as for Example 2, if the recovery of DNA is 574 Q i = 0.3ng/µl, to avoid overloading the PCR reaction, it is necessary to take 575 a dilution of T el = 3µl : T dl = 12µl in order to achieve the optimum 1ng 576 template DNA. Hence, from equation S1: 577 dl = 3 + 12 3 = 5 (S3) With this example, there is a large amount of DNA recovered where 579 Q i = 2ng/µl. The optimum 1ng is therefore contained in just 0.5µl which 580 is difficult to accurately aliquot. Therefore a portion of the eluant is diluted 581 twice in order attain the desired 1ng template for the PCR set-up. The 582 dilution factor is calculated: For the first dilution, an aliquot of T el = 1µl is diluted by the addition 584 of T dl2 = 9µl water,resulting in an estimated 0.2 ng/µl of template. In the 585 second round of dilution we take 1ng template which is in V el = 5µl, added 586 to T dl = 10µl so that a total of 15µl is added to the PCR set-up.

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The dilution factor is calculated from eq. S4: Then the total quantity of DNA recovered is calculated by dividing RF U tot