RiboPure™ RNA Purification Kit, bacteria

Protocol tips

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

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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Manufacturer protocol

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

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