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June 28, 2022 | Microbiome

A glimpse into the microbial RNA world

Tips and tricks to isolate and analyze RNA from microbial samples

Unravelling the microbial world is necessary to better understand microbes’ impacts on our daily lives. However, not all microbes can be cultured in the lab. That’s why it is often easier to get a snapshot of the microbial RNA content of a sample to infer which microbes are present. 

Unfortunately, working with RNA is a bit tricky since this molecule is highly sensitive. So, when processing your microbiome sample, it is incredibly important to protect its RNA from degradation to acquire a reliable snapshot.

Our webinar “Tips and tricks for the isolation and analysis of RNA from microbial samples” gives you practical advice on how to handle and process your sample to improve your results, reproducibility and lab routine. The experts of the webinar stress how important it is to prevent the contamination of your sample. You definitely want to avoid introducing RNases that might otherwise break down the sample’s RNA.

Also, the lysis method is crucial to gain access to the RNA of the microbial cells. For this, our experts highly recommend the mechanical disruption based on the bead beating technology for efficient homogenisation of your sample. However, this also leads to the exposure of molecules that can inhibit downstream applications like PCR or Next-Generation Sequencing (NGS). To get rid of these inhibitors from your sample, all our Power kits rely on the Inhibitor Removal Technology. 

In the next step, you will need to apply the sample to a silica membrane so that the RNA binds to the column material while the rest of the sample is being discarded. The bound RNA is then being washed and finally eluted.

To gain more insights into the microbial workflow as well as practical tips to process your sample, watch the recording of the webinar. Also, each sample type comes with its own challenge. Our experts give valuable ideas and practical advice on how to handle different sample types and extract the RNA to get a glimpse into their microbial communities.

Are you not sure which of our kits is the most suitable for your microbiome sample? Use our selection guides for human microbiome samples and environmental samples to find the best option for your research question.

Also, check out our FAQs about DNA isolation from microbial samples.

Top 25 of our most asked questions about RNA isolation from microbial samples

Sample disruption
  • Bead beating produces heat. Can that compromise RNA integrity?

Yes, bead beating produces small amounts of heat in the process. We suggest to follow the protocol and not to extend the bead beating step for longer than the recommended time to avoid extensice heat development. If you think a longer bead beating step is necessary for your sample to fully disrupt its content, you can put the tubes on ice in between the beating steps.

  • Can I disrupt my samples without a vortex?

Yes, you can use the TissueLyser II instrument to help you with the bead beating step.

  • Is it possible to reuse the beads for purifications?

We recommend not to reuse the beads to avoid cross-contamination. They are difficult to clean, so it is best to use new tubes for different samples. 

  • To homogenise my sample, what is more effective: the bead beating step or manual processing with a mortar and pestle?

The automated bead beating step on the instruments provide much more force to efficiently disrupt your sample. The instruments further provide consistency of the homogenisation process, while you can also choose the specific speed and time. This is especially important when having to disrupt multiple samples and assuring consistent sample processing.

Sample processing
  • After extracting the RNA, can we freeze the sample directly or should I wait for a bit?

We have no data on whether immediate freezing of the sample or after a certain amount of time has an influence on its content.

  • How long can RNA survive on the bench?

Not for longer than 30 minutes! Always store your RNA samples at least at -20 °C.

  • How many freeze-thaw cycles can I do to my sample before it affects the sample’s RNA results?

We have no data on this question but we recommend not more than 5 cycles.

  • How long can I keep RNA in the freezer before testing it?

If you store it at -20 °C, don’t keep it for more than a few months. If you keep your sample at -70 °C, the RNA will remain stable for much longer.

  • Should I keep my RNA samples on ice for downstream applications?

This is up to you. For some, RNA needs to stay on ice all the time. For other, RNA is also stable enough at room temperature.

  • How can I get good RNA samples in consistent replicates?

Make sure to efficiently homogenise your sample. Do not overload your experiment and process only the amount of samples that you can handle. For example, limit your sample number by the spaces in your centrifuge so you don’t have to leave samples standing on the bench.

  • If I flash-freeze my stool samples with liquid nitrogen, how long can I keep them at -80 °C?

Frozen samples at -80 °C are stable for long periods of time. However, avoid any thawing-freezing cycles so that your sample does not get degraded.

RNA extraction from specific sample types
  • How should stool samples be stored prior to RNA extraction?

You can store your stool samples in the PowerProtect DNA/RNA to stabilize your microbial communities. Otherwise, freezing the samples is an option but often leads to rapid degradation of nucleic acids during the thawing process.

  • Can I use your extraction method to recover fungi from stool samples?

To isolate fungal DNA, we recommend the QIAamp PowerFecal Pro DNA Kit; for fungal RNA the RNeasy PowerFecal Pro and for both nucleic acids from one sample you can use the AllPrep PowerFecal Pro DNA/RNA kit.

  • How can I easily extract RNA from sputum samples?

Add 1 volume of Sputal or PBS and incubate the sample at 37 °C until the sample is completely liquefied. Pellet the cells and transfer them to the bead beating tubes. Proceed with the protocol you chose e.g. RNeasy PowerFecal Pro.

  • When sampling skin swabs, we have low yields of RNA. Do you have any recommendations to increase RNA yields from this sample type?

Add the swab directly to the tube containing the beads, for which we recommend the Power Bead Pro Tubes. Otherwise, it is tricky to detach the microbes from the swabs. After the bead beating step and the centrifugation step, your microbial content will remain in the supernatant of the sample.

  • How can I extract RNA from biofilm samples?

We offer a solution to isolate RNA from biofilms and mats with our RNeasy PowerBiofilm Kit. This also includes the bead beating and IRT steps.

  • I want to extract RNA from soil with high-clay content. What do you recommend for me to do?

The best option will be the RNeasy PowerSoil Total RNA Kit. Since the concentration of microbes in this sample type is likely to be low, we recommend higher a sample volume. This kit can handle up to 2 g of soil.

  • What are your recommendations to extract bacterial DNA and RNA from rocks?

You can either swab the rock and use the swab as the sample material and process it directly. Another option would be to crush the rocks with mortar and pestle and process the pieces similar to soil samples with the DNeasy PowerSoil Pro or RNeasy PowerSoil Total RNA Kits.

  • I am using the RNeasy PowerSoil Total RNA Kit for iron-rich mats; samples that are quite rocky. How can we increase the yields for our samples?

Since this sample type contains low biomass to work with, we recommend increasing the sample weight up to 2 g to increase the RNA content of the sample.

  • What is the best way to isolate RNA from sludge samples?

Sludge samples can be compared to wastewater. Hence, we recommend processing up to 40 ml of sludge sample and process the pellet with our RNeasy PowerFecal Pro Kit.

  • Do you have any recommendations to isolate RNA from fruits?

You can use our RNeasy PowerPlant Kit for this sample type.

  • Do you have solutions to isolate microbial RNA from small insects, like mosquitoes?

To isolate RNA from this sample type, we recommend using the RNeasy PowerFecal Pro Kit. The included bead beating step will also efficiently disrupt mosquito samples.

  • Do you have a solution to extract both DNA and RNA from mosquitoes for NGS?

We recommend the AllPrep PowerFecal Pro DNA/RNA kit to extract both DNA and RNA from your sample in separate eluates. You start with one sample and first let DNA bind to a column and elute it. You can then use the flow through to let the RNA bind to a second column to collect it.

  • How can I extract RNA from microbes on cheese?

For such a fatty sample, you can use the RNeasy PowerFecal Pro Kit and add 100 µl Buffer ATL to Solution CD1.

  • What do you recommend for RNA isolation from fungi out of trees?

This depends on which part you are looking at. If you want to isolate fungal cells from leaves, we recommend our RNeasy PowerPlant Kit. If you want to isolate RNA from a pure fungal sample, you can use the RNeasy PowerFecal Pro Kit.