RNeasy Mini Kit

RNA isolation / purification Bacteria - Gram negative Borrelia burgdorferi

Experiment
RNA isolation / purification Bacteria - Gram negative Borrelia burgdorferi
Product
RNeasy Mini Kit from Qiagen
Manufacturer
Qiagen

Protocol tips

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

RNA isolation and qRT-PCR were performed as previously described [29]–[31], [71]. Briefly, when total RNA was isolated from B. burgdorferi by using Trizol (Invitrogen), RNase-free DNase I (GenHunter Corporation) was used to digest genomic DNA. After RNA was further purified using RNeasy Mini Kit (Qiagen), 1 µg of RNA was used to synthesize cDNA using the SuperScript III Platinum Two-step qRT-PCR kit according to the manufacturer’s protocol (Invitrogen). qRT-PCR was employed to examine gene expression, using the relative quantification method (ΔΔCT) as described [29]–[31], [71]. Gene expression fold change was presented as mean ± SE values from three independent experiments. Statistical analyses of the data were performed using the Student’s t test

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Discussion

Discussion

4 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 negative Borrelia burgdorferi using RNeasy Mini Kit from Qiagen.

Paper title
Synthesis of RpoS Is Dependent on a Putative Enhancer Binding Protein Rrp2 in
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Manufacturer protocol

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

Download PDF Download manufacturer protocol

Videos

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