TRIzol Reagent
RNA isolation / purification Bacteria - Gram negative Vibro parahaemolyticus
Protocol tips
| Protocol tips |
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| For low 260/230 readings the best approach is to try washing sample (precipitation) with ethanol to desalt it. |
Publication protocol
Total RNAs were extracted using the TRIzol Reagent (Invitrogen). RNA quality was monitored by agarose gel electrophoresis, and RNA quantity was determined by spectrophotometry. The contaminated DNA in the RNA samples was removed by using the Amibion's DNA-free™ Kit. cDNAs were generated by using 5 μg of RNA and 3 μg of random hexamer primers. Real-time PCR was performed through the LightCycler system (Roche) together with the SYBR Green master mix (Zhan et al., 2008). Based on the standard curve of 16S rRNA expression for each RNA preparation, the relative mRNA levels were determined with the ΔCt values by the classic ΔCt method. 16S rRNA gene was used to normalize.Full paper Login or join for free to view the full paper.
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Discussion
Discussion
5 years ago
Author:
Paul G. Macon
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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Manufacturer protocol
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