RNeasy Mini Kit

RNA isolation / purification Bacteria - Gram negative Escherichia coli

Experiment
RNA isolation / purification Bacteria - Gram negative Escherichia coli
Product
RNeasy Mini Kit from Qiagen
Manufacturer
Qiagen

Protocol tips

Protocol tips
To yield high RNA, give the column an extra wash with both RW1 and RPE buffers (3 total washes with each buffer).
Downstream tips
Include DNAse treatment for 15-20min.

Ensure EtOH is completely evaporated off of the column prior to elution. Adjust time from 1min to 5 min at 60`C.

Use water to elute the RNA that is warmed to ~60`C

Publication protocol

Total RNA was isolated from E. coli cells using a Qiagen RNeasy kit as described by the manufacturer. To determine the integrity of the total RNA samples, 16S and 23S rRNA were resolved by electrophoresis in a 1.2% agarose gel in 1× MOPS buffer containing formaldehyde (20 mm MOPS, 8 mm sodium acetate anhydrous, and 1 mm EDTA, pH 7.0, and 1% formaldehyde). After electrophoresis, agarose gels were stained with ethidium bromide, destained, and photographed under UV light. cDNA was synthesized from total RNA samples using a ThermoScript RT-PCR system. 2.76 μg of RNA was mixed with random hexamer primers (50 ng/μl) and four dNTPs (final concentration, 1 mm). The mixtures were incubated at 65 °C for 5 min and transferred on ice for another 5 min to remove secondary structures of RNA. The denatured RNA samples were then mixed with 1× cDNA synthesis buffer with a total volume of 20 μl containing 5 mm DTT, 40 units of RNaseOut, and 15 units of ThermoScript reverse transcriptase and incubated at 25 °C for 10 min followed by 50 °C for 50 min to synthesize cDNA. The cDNA synthesis mixtures were transferred to an 85 °C water bath for 5 min to terminate the reactions. After the synthesis step, the reaction mixtures were added with 2 units of RNase H and incubated at 37 °C for 20 min to remove the RNA templates.



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Discussion

Discussion

2 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

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Manufacturer protocol

Download the product protocol from Qiagen for RNeasy Mini Kit below.

Download PDF Download manufacturer protocol

Videos

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