RiboPure™ RNA Purification Kit, bacteria

RNA isolation / purification Bacteria - Gram positive Staphylococcus aureus

4/5 - 2 Reviews - 50% recommends this

Experiment
RNA isolation / purification Bacteria - Gram positive Staphylococcus aureus
Product
RiboPure™ RNA Purification Kit, bacteria from Thermo Fisher Scientific
Manufacturer
Thermo Fisher Scientific

Protocol tips

Upstream tips
- Use of physical disruption of cell walls
- Enzymatic digestion of cell walls
Downstream tips
When checking the yield by means of a specrtophotometer, use the supplied TE buffer to zero the machine

Publication protocol

Gene expression analysis was performed on 1-, 3-, and 24-h-old biofilms. Biofilms were grown by adding to each sample 2 ml of the main culture diluted 1:100 with growth medium. Total RNA from the biofilms was isolated using a RiboPure Bacteria kit (Ambion; Invitrogen) according to the manufacturer's instructions. Traces of genomic DNA was removed using a DNA-free kit (Ambion; Applied Biosystems, Foster City, CA), and the absence of genomic DNA contamination was verified by real-time PCR prior to cDNA synthesis. cDNA synthesis was carried out using 200 ng of RNA, 4 μl 5× iScript reaction mix, and 1 μl iScript reverse transcriptase in a total volume of 20 μl (Iscript; Bio-Rad, Hercules, CA) according to the manufacturer's instructions. Real-time reverse transcription-quantitative PCR (qPCR) was performed in triplicate in a 96-well plate (AB0900; Thermo Scientific, United Kingdom) with the primer sets specific for gyrB, icaA, and cidA (Table 1). The following thermal conditions were used for all qPCRs: 95°C for 15 min and 40 cycles of 95°C for 15 s and 60°C for 20 s. The mRNA levels were quantified in relation to the level of the endogenous control gene gyrB. The levels of icaA and cidA expression in all biofilms were expressed relative to their levels of expression in biofilms grown on PE.

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Reviews

Scientist
Review date

Protocol
Standard

Product rating
Would recommend?
No

What worked well?
  • grest
What could be improved?
  • fsdsd

Scientist
Akshay K. Harapanahalli
Review date

Protocol
Standard
Protocol tips
Use of physical disruption of cell walls gives better results.

Product rating
Would recommend?
Yes

What worked well?
  • Cell wall disruption is good for gram positive bacteria
  • Heigh yields of pure RNA for downstream processing
What could be improved?
  • Lengthy protocol

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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 Staphylococcus aureus using RiboPure™ RNA Purification Kit, bacteria from Thermo Fisher Scientific.

Paper title
Influence of Adhesion Force on and Gene Expression and Production of Matrix Components in Biofilms
Full paper
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Manufacturer protocol

Download the product protocol from Thermo Fisher Scientific for RiboPure™ RNA Purification Kit, bacteria below.

Download PDF Download manufacturer protocol

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