RNA isolation / purification Bacteria - Gram negative Helicobacter pylori

Generally isolating RNA from Gram-negative bacteria is easy, however keeping your working environment clean and RNase free (use RNase inhibitor) is essential. Some common points to keep in mind: a) Use fresh samples for isolation or store them by freezing in RNA stabilizing buffer until use. b) Choose the bacterial input amounts carefully, to ensure buffer volumes are adequate and not to overload the columns.

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Discussion

5 years ago

5 years ago by 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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Found 3 matching solutions for this experiment

TRI Reagent® Sigma

Sigma-Aldrich

Upstream tips	Protocol tips	Downstream tips
	Helicobacter pylori strains (Table Table11) were grown with gentle agitation (120 rpm) in 30 ml of Brucella broth at 37°C until mid-exponential phase (OD = 0.7). For heat-shock treatment, the wild type (WT) culture was split into 15 ml-aliquots and one sample was subjected to heat-shock at 42°C for 30 min (heat-shock sample, HS). A volume of 10 ml cell culture was then added to 1.25 ml of ice-cold EtOH-phenol stop solution (5% acid phenol, in EtOH) to stop growth and prevent RNA degradation. Cells were pelleted, stored at −80°C, and then used to extract total RNA with TRI-reagent (Sigma-Aldrich), according to manufacturer’s protocol.

Protocol tips
Helicobacter pylori strains (Table Table11) were grown with gentle agitation (120 rpm) in 30 ml of Brucella broth at 37°C until mid-exponential phase (OD = 0.7). For heat-shock treatment, the wild type (WT) culture was split into 15 ml-aliquots and one sample was subjected to heat-shock at 42°C for 30 min (heat-shock sample, HS). A volume of 10 ml cell culture was then added to 1.25 ml of ice-cold EtOH-phenol stop solution (5% acid phenol, in EtOH) to stop growth and prevent RNA degradation. Cells were pelleted, stored at −80°C, and then used to extract total RNA with TRI-reagent (Sigma-Aldrich), according to manufacturer’s protocol.

Protocol tips

Helicobacter pylori strains (Table Table11) were grown with gentle agitation (120 rpm) in 30 ml of Brucella broth at 37°C until mid-exponential phase (OD = 0.7). For heat-shock treatment, the wild type (WT) culture was split into 15 ml-aliquots and one sample was subjected to heat-shock at 42°C for 30 min (heat-shock sample, HS). A volume of 10 ml cell culture was then added to 1.25 ml of ice-cold EtOH-phenol stop solution (5% acid phenol, in EtOH) to stop growth and prevent RNA degradation. Cells were pelleted, stored at −80°C, and then used to extract total RNA with TRI-reagent (Sigma-Aldrich), according to manufacturer’s protocol.

Manufacturer protocol Publication protocol 1 Relevant paper

SV Total RNA Isolation System

Promega

Upstream tips	Protocol tips	Downstream tips
	Do not freeze-thaw the DNase more than three times after rehydration. After adding ethanol to the RNA wash solution, label the bottle so that you fellow researchers don't add any extra.	For better lysis, pipet the RNA Lysis Buffer over the bottom of the well.

Protocol tips
Do not freeze-thaw the DNase more than three times after rehydration. After adding ethanol to the RNA wash solution, label the bottle so that you fellow researchers don't add any extra.
Downstream tips
For better lysis, pipet the RNA Lysis Buffer over the bottom of the well.

Manufacturer protocol Publication protocol 1 Relevant paper

TRIzol Reagent

Thermo Fisher Scientific

Upstream tips	Protocol tips	Downstream tips
	For low 260/230 readings the best approach is to try washing sample (precipitation) with ethanol to desalt it.

Protocol tips
For low 260/230 readings the best approach is to try washing sample (precipitation) with ethanol to desalt it.

Manufacturer protocol Publication protocol 1 video 1 Relevant paper

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