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P044-NF2
Neurofibromatosis type 2 (NF2)
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P199-HEXA
Tay-sachs disease
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P214-COL2A1
Skeletal disorders, Achondrogenesis, Chondrodysplasia, Early onset familial Osteoarthritis, SED congenital, Langer-Saldino achondrogenesis, Kniest dysplasia, Stickler syndrome, Spondyloepimetaphyseal
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P280-SLC26A4
Pendred syndrome
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P026-Sotos
Sotos syndrome
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ME011-MMR
Mismatch repair genes (MMR)
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mRNA MLPA® (RT-MLPA)
Reverse transcriptase MLPA (RT-MLPA) is a variation on MLPA developed especially for mRNA profiling. Changes in gene expression play an important role in cell development, differentiation and pathological processes. Currently, SALSA RT-MLPA kits are available for the mRNA detection of various apoptosis and inflammation genes.
RT-MLPA offers various advantages compared to other expression profiling techniques such as northern blotting, real time PCR and microarrays. Firstly, it allows the quick processing of numerous samples in a standard PCR 96-well format. Secondly, RT-MLPA analysis is straightforward and yields quantitative information on a medium-sized gene (1). Although the preferred amount of sample RNA is 20 to 200 ng, RT-MLPA has been used successfully on as little as 5-10 ng.
The MLPA Ligase-65 enzyme cannot ligate DNA oligos which are annealed to an RNA target. To circumvent this, RT-MLPA starts with a reverse transcriptase reaction (Figure 1). Each SALSA RT-MLPA kit comes with specifically designed RT-primers, which anneal directly adjacent to or even overlap with the probe recognition site, thus reverse transcribing short mRNA fragments into cDNA. As short cDNA fragments are already sufficient for the probes to bind, the influence of RNA degradation on RT-MLPA results is small. One can even use RNA extracted from paraffin-embedded, formaldehyde-treated tissues, although success rates will strongly depend on the tissue’s conditions (e.g. pH-level, age).
After the RT reaction, RT-MLPA continues like a standard MLPA, starting with hybridization of probes to their target cDNA. Whenever possible, RT-MLPA probes are designed to included an exon boundary in their target sequence: one part of a probe may hybridize to the last 25 nucleotides of exon 1, while the other binds to the first 35 nucleotides of exon 2. Such an “intron spanning” design prevents the probe from generating a signal on contaminating genomic DNA that is often present in RNA samples.
Figure 1
References
- Eldering, E., Spek, C.A., Aberson, H.L., Grummels, A., Derks, I.A., de Vos, A.F., McElgunn, C.J. and Schouten, J.P. (2003). Expression profiling via novel multiplex assay allows rapid assessment of gene regulation in defined signalling pathways Nucleic Acids Res 31, e153.
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