TRIsure™

RNA isolation / purification Tissue - Human Choroid plexus

Experiment
RNA isolation / purification Tissue - Human Choroid plexus
Product
TRIsure™ from Bioline
Manufacturer
Bioline

Protocol tips

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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Discussion

Discussion

4 years ago

Author: Paul G. Macon United States

RNA isolation from tissue

How do I extract RNA from animal tissue without using liquid nitrogen? I tried the RNA extraction by using the TRIzol reagent and I homogenize the tissue using polytron homogenizer at room temperature for 30secs is this correct?

Discussion

5 years ago

Author: Aaron Stege Netherlands

Problem in phase separation after using serum/plasma kit

I used a serum/plasma kit for my serum samples. After the phase separation the samples should have 3 phases: a colourless aqueous phase, a white interphase and a red organic phase. However, in some of my samples there was no aqueous phase unless I wait for an extended period of time. How can I circumvent this problem?

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Papers

Check out relevant papers found by Labettor's AI that are relevant for performing RNA isolation / purification Tissue - Human Choroid plexus using TRIsure™ from Bioline.

Paper title
Isolation of primary microglia from the human post-mortem brain: effects of ante- and post-mortem variables
Full paper
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Manufacturer protocol

Download the product protocol from Bioline for TRIsure™ below.

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Videos

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