miRNeasy Mini kit

RNA isolation / purification Bacteria - Gram positive Listeria monocytogens

Experiment
RNA isolation / purification Bacteria - Gram positive Listeria monocytogens
Product
miRNeasy Mini kit from Qiagen
Manufacturer
Qiagen

Protocol tips

Protocol tips
- To capture a higher amount of mRNA, modify the amount of lysis buffer :ethanol (from 1:1 to 1:1.5) during the binding step.

Publication protocol

Total RNA was extracted using miRNeasy kit (Qiagen) with some modifications. The collected pellets were washed with SET buffer [50 mM NaCl, 5 mM EDTA and 30 mM Tris-HCl (pH 7.0)]. After centrifugation at 16000×g for 3 min pellets were resuspended in 0.1 ml Tris-HCl (pH 6.5) containing 50 mg/ml lysozyme (Sigma), 25 U of mutanolysin (Sigma), 40 U of SUPERase (Ambion), 0.2 mg of proteinase K (Ambion) and incubated at 37°C for 30 min at 350 rpm. QIAzol (Qiagen) was added, mixed gently and incubated for 3 min at room temperature. An additional incubation at room temperature was done after adding 0.2 volume chloroform followed by centrifugation at 16000×g at 4°C for 15 min. The aqueous phase, containing RNA, was transferred to a new collection tube and 1.5 volumes of 100% ethanol was added and mixed thoroughly. The probes containing RNA were transferred into columns supplied with the miRNeasy Kit (Qiagen) and treated according to the manual including an on-column DNase digestion (RNase-Free DNase, Qiagen). RNA was eluted by RNase-free water and stored at −80°C until needed. The quantity of the isolated total RNA was determined by absorbance at 260 nm and 280 nm, and the quality was assessed using Nano-chips for Agilent's 2100 Bioanalyzer. For detection and estimation of the small RNA fraction within the isolated total RNA, a small RNA-chip (Agilent) was used, which visualizes RNAs with sizes ranging from 20 to 150 nucleotides.

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Discussion

Discussion

5 years ago

Author: Paul G. Macon United States

Some help with RNA isolation using Trizol

Hello! I used Trizol to extract total RNA from in-vitro cultured bacteria (1 X 10^8 cells). After phase separation, I mixed ~0.4 ml of the upper phase which contains RNA with 0.5 mL cold isopropanol. However, the amount of RNA when measured in Nanodrop was very low. In addition, the ratio between 260 and 230 was around 0.1 to 0.5. Is there a chance that my sample was contaminated by the Trizol reagent? When I collected the aqueous phase I made sure to not touch the lower phase. What should I do?

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Papers

Check out relevant papers found by Labettor's AI that are relevant for performing RNA isolation / purification Bacteria - Gram positive Listeria monocytogens using miRNeasy Mini kit from Qiagen.

Paper title
Ultra Deep Sequencing of sRNA Transcriptome Revealed New Antisense RNAs
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Manufacturer protocol

Download the product protocol from Qiagen for miRNeasy Mini kit below.

Download PDF Download manufacturer protocol

Videos

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