QuantiTect Reverse Transcription Kit

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	For Genomic DNA elimination, incubate reactions for 2 min at 42°C, then place immediately on ice. Add Reverse-transcription reaction components and incubate for 15 min at 42°C. Reverse-transcription reaction components

Protocol tips
For Genomic DNA elimination, incubate reactions for 2 min at 42°C, then place immediately on ice. Add Reverse-transcription reaction components and incubate for 15 min at 42°C. Reverse-transcription reaction components

Publication protocol

For RNA extraction, brain tissue samples were lysed with RLT-buffer (Qiagen, Hilden, Germany) and homogenized with a MM300 mill mixer (Retsch, Haan, Germany) at 30 Hz for 2 minutes. Then, we isolated the total RNA from the lysed and homogenized cells by using a RNeasy Lipid Tissue Mini Kit (Qiagen) according to the manufacturer's instructions and eluted these with 30 μL to 50 μL RNase-free water. RNA quality was confirmed via gel electrophoresis (Experion Automated Electrophoresis System, Bio-Rad, Munich, Germany) for RQI (RNA quality indicator) ≥8 [11]. We calculated the RNA concentration, using a Quanti-iT™ RNA Assay kit with the Qubit™ fluorimeter (Molecular Probes™, Thermo-Fisher Scientific, Germany) and by spectral absorption (A260 nm/A280 nm; NanoVue, General Electrics, MA, USA). Most scientist worldwide use the A260/A280 absorption method to check for e.g. protein contamination and purity of the RNA with a ratio of ~2.0 generally accepted as “pure RNA”. Abnormal 260/280 ratios usually indicate contamination by proteins or reagents such as phenol. DNA/RNA shreds and single nucleotides also contribute to the absorption at A260 and may serve as confounding factor. To overcome this limitation specific assays were developed with RNA-binding dyes. The Quant-iT™ RNA assay was designed to specifically quantify RNA, rRNA, or mRNA in a range from 250 pg/μL to 100 ng/μL (excitation/emission maxima 644/673 nm). Unlike UV absorbance measurements at 260 nm, the reagent does not detect significant sample contamination by free nucleotides. Thus, the Quant-iT™ RNA assay more accurately measures the amount of intact RNA polymers in potentially degraded samples. Unfortunately, detailed information on dye, specificity for RNA, and performance compared with other dyes, e.g. RiboGreen [12], are not available for the general public due to strict confidentiality policy of the manufacturer.

Quantitative polymerase chain reaction (real-time PCR)
We amplified equal amounts of cDNA (1 μL) of each sample in triplicates, using the real-time Lightcycler 480 PCR System (Roche). Table 2 shows the real-time cycling parameters and applied primers and probes. We used mRNA-specific primers for real-time PCR. Each run was designed to contain samples of all points in time and regions to minimize the influence of run-to-run variations. We performed a total of six runs for each gene product.

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Manufacturer protocol

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