Additional file 7: of Identification of TCERG1 as a new genetic modulator of TDP-43 production in Drosophila

Figure S5. Quantification of TDP-43 steady-state mRNA levels by RT-QMPSF. (A) Schematic representation of the TDP-43 transcription unit and the relative location of the RT-QMPSF amplicons. (B) Expression analyses of TDP-43 mRNA transcripts by RT-QMPSF. This assay is based on simultaneous PCR amplification of short fluorescent fragments in a single tube. The single-stranded cDNA was PCR-amplified using: TDP-43F1/R1 yielding a 115 bp product, TDP-43F2/R2 that yielded fragments of 132 bp and CG42724 that produced an amplicon of 173 bp (Additional file 1: Figure S1A). RpL13A (141 bp) and Cyp1 (162 bp) cDNAs were amplified as internal references. The number of cycles of amplification was determined by testing a range of cycle numbers in order to remain in the linear phase of the PCR. Fluorescent amplicons were separated on a genetic analyzer and the resulting fluorescent profiles were analyzed. The diagrams shown were obtained from GMR > + (control, blue), GMR > TDP-43_TDPBR (red) or GMR > TDP-43_TDPBR, UY5237 (green) flies. The y-axis displays fluorescence in arbitrary units, and the x-axis indicates the size in bp. The electropherograms were superimposed by adjusting the peaks obtained for the control amplicons to the same level. (C) TDP-43 amplicons were amplified using a TDP-43-specific primer (F3) and an oligo-dT adapter primer (AUAP). The diagrams shown were obtained from GMR > + (control, blue), GMR > TDP-43_TDPBR (red) or GMR > TDP-43_TDPBR, UY5237 (green) flies. Cyp1 cDNAs was amplified as internal reference. The electropherograms were superimposed by adjusting the peaks obtained for the control amplicons to the same level. Note that the “mis-alignement » of the longest pics is due to the imprecise sizing of the fragment > 1 kb. (TIF 1151 kb)