RNeasy Mini Kit

RNA isolation / purification Bacteria - Gram positive Streptococcus pneumoniae

Experiment

RNA isolation / purification Bacteria - Gram positive Streptococcus pneumoniae

Product

RNeasy Mini Kit from Qiagen

Manufacturer

Qiagen

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Tips Publication protocol Reviews Discussion Papers Manufacturer protocol Videos

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	To yield high RNA, give the column an extra wash with both RW1 and RPE buffers (3 total washes with each buffer).	"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"

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

Once thawed, NP samples (200 µl) had 1 volume of RNAprotect Bacteria® (Qiagen) added and were immediately centrifuged for 15 min at 15000×g in a refrigerated centrifuge (Eppendorf). Total RNA was then extracted from the pellet using the RNeasy Mini Kit (Qiagen) as outlined by the manufacturer and additionally treated with 2 U of DNaseI (Promega) essentially as previously described [31], [40]. Integrity of our RNA preparations [RNA integrity number (RIN)], and RNA concentration of samples, were obtained by using the RNA 6000 Nano kit or RNA 6000 Pico kit electrophoresis system and the 2100 Bioanalyzer (Agilent technologies). Total RNA (500 pg) was cDNA transcribed using the iScript cDNA synthesis kit (Bio-Rad) and following the manufacturer's instructions.

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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 Streptococcus pneumoniae using RNeasy Mini Kit from Qiagen.

Paper title

Expression of Virulence-Related Genes in the Nasopharynx of Healthy Children

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Manufacturer protocol

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

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Videos

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