MRC-Holland - MLPA Procedure

	introduction
	experimental set up
	sample treatment
	MLPA reaction
	fragment separation
	raw data evaluation
	peak pattern evaluation
	data analysis
	result interpretation
	troubleshooting

new products
P339-SHANK3 Autism Spectrum Disorder
P010-POLG Mitochondrial maintenance
P366-CHM-RP2-RPGR Retinitis pigmentosa (RP)
P350-CLCN1-KCNJ2 Myotonia congenita, Thomsen’s disease, Becker’s disease, Andersen-Tawil syndrome
P273-PCSK5 Atherosclerotic cardiovascular disease (risk of)

improved products
P044-NF2 Neurofibromatosis type 2 (NF2)
P199-HEXA Tay-sachs disease
P214-COL2A1 Skeletal disorders, Achondrogenesis, Chondrodysplasia, Early onset familial Osteoarthritis, SED congenital, Langer-Saldino achondrogenesis, Kniest dysplasia, Stickler syndrome, Spondyloepimetaphyseal
P280-SLC26A4 Pendred syndrome
P026-Sotos Sotos syndrome
ME011-MMR Mismatch repair genes (MMR)

The MLPA^® procedure from A to Z

The quality of your MLPA results does not only depend on a reliable data normalisation method, but also on the design of the experimental setup (the choice of test and references samples), on how the MLPA protocol is performed and on instrument settings. In this section, you can find everything you need to know about the MLPA procedure.

The MLPA procedure consists of eight interrelated steps (see below). In case earlier steps are not performed properly, later steps may fail. To obtain good results, control checks should be included and verified in each MLPA step. If you get unclear results, please see our troubleshooting section for support.

The MLPA Procedure in eight steps

1) Experimental set up
Reference samples are generally essential for MLPA ratio calculations. Please follow these recommendations on the use of control and reference samples.

2) Sample treatment
Sample preparation, such as the DNA extraction, can influence MLPA results. In this section you can learn more on how to treat your samples prior to the MLPA reaction.

3) MLPA reaction
MLPA protocols contain several suggestions on how to minimise variation in the MLPA procedure and thus optimise results.

4) Fragment separation
Optimal capillary fragment separation settings differ between machines. In this section, you can learn how to establish the optimal fragment separation settings for your device.

5) Raw data evaluation
Raw data should be evaluated according to the raw data checklist. This checklist will allow you to quickly detect any problems caused by the fragment separation device.

6) Peak pattern evaluation
The MLPA Evaluation & Troubleshooting Flowchart will help you locate failures of the MLPA reaction or fragment separation. Only electropherograms passing this flowchart should be used for data analysis!

7) Data analysis
How to process your raw data to obtain reliable copy number ratios.

8) Result interpretation
How to interpret your calculated ratios and what they tell you about the samples studied.

Troubleshooting
If you follow the above guidelines and verify each step, reliable MLPA results will be obtained. In case you are experiencing problems however, this section will help you to locate and solve them.

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