The main sample requirements for conventional MLPA and digitalMLPA are listed below.
A conventional MLPA reaction requires a total quantity of 50–250 ng of DNA in a 5 µl volume (50–100 ng is optimal). A digitalMLPA reaction requires 20–250 ng in a 4 µl volume (40–100 ng is optimal). The instructions for some products may differ.
The DNA sample volume should never be increased above the stated volume (5 µl for conventional MLPA; 4 µl for digitalMLPA). This reduces the concentrations of probes and salts, which in turn negatively affects the hybridisation efficiency and stability.
DNA concentration measurements based on optical density (260 nm) tend to overestimate DNA concentrations, e.g. due to the presence of RNA. Whether sufficient DNA was added can be estimated based on the signals of the Q-fragments in a conventional MLPA experiment. For more information about the Q-fragments and how to interpret them, see this e-learning module. Coffalyser.Net™ for conventional MLPA and Coffalyser digitalMLPA™ for digitalMLPA will provide a warning if there was insufficient sample DNA present.
We recommend dissolving and diluting samples in TE0.1 (10 mM Tris-HCl pH 8.0 + 0.1 mM EDTA).
It is important that the DNA sample contains 5–10 mM Tris-HCl buffer with a pH of 8.0–8.5 to prevent depurination. For conventional MLPA experiments, we recommend adding 1 µl of 50 mM Tris-HCl pH 8.5 to 4 µl of sample DNA if it is unknown if sufficient buffering capacity is present.
Higher concentrations of EDTA in the TE buffer should be avoided, as this may lead to reaction failure. Conventional MLPA experiments are more sensitive to the EDTA concentration than digitalMLPA experiments.
Never dissolve or dilute samples in water, as it lacks sufficient buffering capacity. In addition, do not use PCR buffer, as it contains salts that can hamper denaturation.
Suitable DNA sample origin varies between probemixes. Always consult the relevant instructions for use for suitable tissue sources. Examples of tissue sources that are suitable for some products are: blood, buccal swabs, fresh healthy/tumour tissue, or FFPE healthy/tumour tissue.
Samples should be free from impurities known to affect (digital)MLPA reactions. See this article for more details.