As an example, the dispersion of 459 pg of sample DNA (containing 70 copies of a SNP allele from a heterozygous genotype) across the 765 chambers of a panel results in most chambers being unoccupied with target DNA sequence, but those 70 copies present are mostly isolated in individual reaction chambers.
To quantify HPV58 and host GAPDH copy numbers from the same sample before and after RCA, a quantitative real-time PCR (qPCR) was performed on ~100 pg of sample DNA (samples 10 and 13).
cDNA samples were quantified using the Fluorescent DNA Quantitation Kit (Bio-Rad, UK) according to manufacturer's specifications, and 250 pg of cDNA sample was used per reaction.
This generated a concentration gradient of specific Lucidea transcripts of 0.1, 0.3, 1, 3, 10, 30, 100, 1000 and 3000 pg per μg of sample total RNA.
Despite the fact that the low-affinity binding of HMGB1 to single-strand DNA results in a decrease of the signal (as observed when comparing the equivalent calibration samples in Figs. 1, 2, 3), under such conditions it is possible to detect 10 pg of HMGB1 per sample (Figure 3A).
As for bioanalyzer analysis, 500 pg of DNA per sample was loaded on the chip and analyzed on Agilent 2100 Bioanalyzer, as per manufacturer's instructions (Agilent Technology, High Sensitivity DNA Kit, 5067 4626).
The sensibility levels were: 10 pg of u-PA/ml of sample; 0.1 ng of u-PAR/ml of sample; 1 ng of PAI-1 /ml of sample.
It was successfully coupled to inductively coupled plasma mass spectrometry (ICP-MS) achieving detection limits below 100 pg kg−1 for 5 300 mg of sample.
A total of 750 pg of RNA from each sample was amplified using Ovation RNA-seq System V2 (NuGEN Technologies, CA).
A cutoff > 940 pg of human DNA per sample was a predictor for a positive fungal DNA PCR (1/7 vs. 28/33; p = 0.001).
The Lucidea RNA mix (4 μl) was added to sample total RNA (20 μg) to produce a concentration gradient of 0.1, 0.3, 1, 3, 10, 30, 100, 1000 and 3000 pg of Lucidea transcripts per μg of sample RNA.
