TRIsure™

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	- To reduce DNA contamination wash pellet with 0.1 M sodium citrate in 10% ethanol. - To increase RNA yield, Increase the solubilization by pipetting the sample repeatedly, heat the sample to 60 °C	- If there is Low A260/280 for RNA,ensure that 1 mL TRIsure per 10 cm2 area of cells or 5 x 10^6 cells is used.

Protocol tips
- To reduce DNA contamination wash pellet with 0.1 M sodium citrate in 10% ethanol. - To increase RNA yield, Increase the solubilization by pipetting the sample repeatedly, heat the sample to 60 °C
Downstream tips
- If there is Low A260/280 for RNA,ensure that 1 mL TRIsure per 10 cm2 area of cells or 5 x 10^6 cells is used.

Publication protocol

Acutely isolated primary microglia were taken up in 1 ml TRIsure (Bioline, London, UK) and stored at −80 °C for further processing. RNA isolation was carried out according to manufacturer’s protocol using phase separation by addition of chloroform and centrifugation, followed by overnight precipitation in isopropanol at −20 °C. RNA concentration was measured using a Nanodrop (ND −1000; NanoDrop Technologies, Rockland, DE, USA) and RNA integrity was assessed using a Bioanalyzer (2100; Agilent Technologies, Palo Alto, CA, USA). cDNA synthesis was performed using the Quantitect reverse transcription kit (Qiagen, Hilden, Germany) according to manufacturer’s instructions, with a minimal input of 200 ng total RNA. Quantitative PCR (qPCR) was performed using the 7300 Real Time PCR system (Applied Biosystems, Foster City, USA) using the equivalent cDNA amount of 1–2 ng total RNA used in cDNA synthesis. SYBRgreen mastermix (Applied Biosystems) and a 2 pmol/ml mix of forward and reverse primer sequences were used for 40 cycles of target gene amplification. An overview of forward and reverse sequences for each gene can be found in Additional file 1: Table S1. Expression of target genes was normalized to the average cycle threshold of GAPDH and EF1a. Cycle threshold values were assessed with SDS software (Applied Biosystems).

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Manufacturer protocol

Download the product protocol from Bioline for TRIsure™ below.

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