Faststart universal sybr green

For qRT-PCR analyses, gene-specific oligonucleotide primers were designed (Table 2) and the gene specificity of each pair of primers was checked by melting curves and product re-sequencing twice. The glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene was employed as the internal control for calculating relative expression of the mRNA [56 (link)]. The sequences of GAPDH primers are described in Table 1. qRT-PCR was performed by FastStart Universal SYBR Green (Roche Diagnostics, Shanghai, China), initiated by 10 min at 95 °C and followed by 40 cycles of 95 °C for 30 s, 60 °C for 30 s, and 72 °C for 10 min, and completed with a melting curve analysis program. The PCR mixture (10 μL total volume) comprised 5 μL of Roche FastStart Universal SYBR Green Master (ROX), 0.75 μL of each primer (10 μmol/L), 0.5 μL of diluted cDNA and 3 μL PCR-grade ddH₂O. No-template controls and melting curve analysis were included for each gene during each run.

For qRT-PCR analyses, gene-specific oligonucleotide primers were designed and described in Table 3. The gene specificity of each pair of primers was checked by melting curves and product re-sequencing twice. The GAPDH gene was employed as the internal control for calculating relative expression of the mRNA [53 (link)]. The sequences of GAPDH primers are described in Table 3. Real-time PCR was performed using FastStart Universal SYBR Green (Roche, Basel, Switzerland), initiated by 10 min at 95 °C and followed by 40 cycles of 95 °C for 30 s, 60 °C for 30 s, and then by 72 °C for 10 min, and completed with a melting curve analysis program. The PCR mixture (10 μL total volume) was comprised of 5 μL of Roche FastStart Universal SYBR Green Master (ROX) (Roche, Basel, Switzerland), 0.75 μL of each primer (10 μM), 0.5 μL of diluted cDNA and 3 μL of PCR-grade ddH₂O. No-template controls and melting curve analysis were included for each gene during each run.

For real-time RT-PCR analysis, WT, 5XFAD, APP/PS1KI and APP23 mice (n = 3–6 per group) or RNA extracts from BV2 cells (n = 6) were used. For RNA isolation, deep-frozen brain hemispheres or spinal cord tissue were homogenized in TriFast reagent (Peqlab) essentially as described previously [16 (link)]. Deep frozen liver samples were homogenized in 1 ml TriFast reagent (Peqlab) per 100 mg tissue using a glas-teflon homogenizer. BV2 cell pellets were homogenized manually in 1 ml TriFast reagent by repetitive pipetting. DNAse digestion and reverse transcription of the purified RNA samples were carried out according to the protocol of the manufacturer (Thermo Fisher). RT-PCR was performed using a Stratagene MX3000 Real-time Cycler. The SYBR green based FastStart Universal SYBR Green (Roche) containing ROX as an internal reference dye was used for amplification. Relative expression levels were calculated using the 2^{- ∆∆Ct} method and normalized to the housekeeping gene β-actin [43 (link)]. Primer sequences can be found in Additional file 2.

A 1 mm cross section of graft and abdominal aorta was snap frozen with liquid nitrogen. These snap frozen sections were halved, and RNA was extracted with Trizol reagent (Invitrogen, CA, USA) until the aqueous phase step, followed by the use of RNA extraction columns (Bioline #BIO-52073). RNA (200 ng) was reverse transcribed (25 °C–10 min; 42 °C–15 min; and 85 °C–5 min) using the Bioline SensiFAST™ cDNA Synthesis Kit (Bioline #BIO-65054). A total of 4 ng cDNA were used for quantitative analysis (two-step RT-PCR, 8.5 min at 95 °C; [38 × 15 s at 95 °C; 45 s at 60–63.5 °C]; 1 min at 95 °C) of rat genes (supplementary material Table 1) with FastStart Universal SYBR Green (Roche #4913914001), followed by a melting curve. B2M and GAPDH were used as standard housekeeping genes. The relative mRNA expression levels were determined using the ΔCT method.