To show the mRNA level of app family members, RNA was extracted from whole larvae at 24hpf. Embryos of AB or appb +/+ background were used as controls. Each experiment was performed at least three times with four to five technical replicates (considered as "N") of 10 larvae each. Total RNA was extracted from 10 larvae, using TRI Reagent ® (Sigma Aldrich).
Then, RNA samples were treated with RQ1 1x RNase-free DNase reaction buffer and RQ1 RNase-free DNase (Promega kit). cDNA was synthesized using High-Capacity RNA-to-cDNA Kit (Applied Biosystems) and converted in a single-cycle reaction on a 2720 Thermal Cycler (Applied Biosystems). Quantitative PCR (qPCR) was performed with inventoried TaqMan Gene Expression Assays with FAM reporter dye (Thermo Fisher Scientific) in TaqMan Fast Advanced Master Mix (Applied Biosystems). The assay was carried out on Micro-Amp 96-well optical microtiter plates on a QuantStudio 3 Real-Time PCR System Software (Applied Biosystems). qPCR results were analysed with the QuantStudio Design and Analysis software v1.5.2 (Applied Biosystems). Briefly, CT from each sample was normalized with average CT:s of eef1a1l1 and actb1, then the relative quantity was determined using the ΔΔCT method (Livak & Schmittgen, 2001) (link) with a sample of wildtype embryos (6-72 hpf and adult brain) as the calibrator. TaqMan Gene Expression Assays (Applied Biosystems) were used for the following genes: Amyloid Beta (A4) Precursor Protein A (appa, Dr 03144364_m1 and Dr 03144365_m1), Amyloid Beta (A4) Precursor Protein B (appb, Dr 03080308_m1 and Dr 03080304_m1 ), Amyloid Beta Precursor Like Protein 1 (aplp1, AJCSWD2), Amyloid Beta Precursor Like Protein 2 (aplp2, Dr 03437773_m1), Eukaryotic Translation Elongation Factor 1 Alpha 1, Like 1 (eef1a1l1, Dr 03432748_m1) and Actin Beta 1 (actb1, Dr 03432610_m1). To confirm the deletion of appb in appb P-/-SYBR green primers were designed to amplify exon 16-17 in appb and eef1a1 primer pairs used as housekeeping gene (Extended Dats Table 4-3).
e N e u r o A c c e p t e d M a n u s c r i p t
Then, RNA samples were treated with RQ1 1x RNase-free DNase reaction buffer and RQ1 RNase-free DNase (Promega kit). cDNA was synthesized using High-Capacity RNA-to-cDNA Kit (Applied Biosystems) and converted in a single-cycle reaction on a 2720 Thermal Cycler (Applied Biosystems). Quantitative PCR (qPCR) was performed with inventoried TaqMan Gene Expression Assays with FAM reporter dye (Thermo Fisher Scientific) in TaqMan Fast Advanced Master Mix (Applied Biosystems). The assay was carried out on Micro-Amp 96-well optical microtiter plates on a QuantStudio 3 Real-Time PCR System Software (Applied Biosystems). qPCR results were analysed with the QuantStudio Design and Analysis software v1.5.2 (Applied Biosystems). Briefly, CT from each sample was normalized with average CT:s of eef1a1l1 and actb1, then the relative quantity was determined using the ΔΔCT method (Livak & Schmittgen, 2001) (link) with a sample of wildtype embryos (6-72 hpf and adult brain) as the calibrator. TaqMan Gene Expression Assays (Applied Biosystems) were used for the following genes: Amyloid Beta (A4) Precursor Protein A (appa, Dr 03144364_m1 and Dr 03144365_m1), Amyloid Beta (A4) Precursor Protein B (appb, Dr 03080308_m1 and Dr 03080304_m1 ), Amyloid Beta Precursor Like Protein 1 (aplp1, AJCSWD2), Amyloid Beta Precursor Like Protein 2 (aplp2, Dr 03437773_m1), Eukaryotic Translation Elongation Factor 1 Alpha 1, Like 1 (eef1a1l1, Dr 03432748_m1) and Actin Beta 1 (actb1, Dr 03432610_m1). To confirm the deletion of appb in appb P-/-SYBR green primers were designed to amplify exon 16-17 in appb and eef1a1 primer pairs used as housekeeping gene (Extended Dats Table 4-3).
e N e u r o A c c e p t e d M a n u s c r i p t
