NEB Podcast #82 -
Transcript
Interviewers: Lydia Morrison, Marketing Communications Manager & Podcast Host, New England Biolabs, Inc.
Interviewee: Anagha Kadam, Ph.D., Development Scientist II, New England Biolabs; Jack Martz, M.S., Technical Support Scientist I, New England Biolabs
Lydia Morrison:
Welcome to the Lessons from Lab & Life podcast brought to you by New England Biolabs. I'm your host, Lydia Morrison, and I hope this episode brings you some new perspective. Today, I'm joined by two NEB scientists, Anagha Kadam and Jack Martz who will talk us through their tips to optimize your next RNA isolation.
Anagha and Jack, thanks so much for being here to join me today. Before we get started, could you both introduce yourselves and tell our listeners what it is you do here at New England Biolabs?
Anagha Kadam:
Sure. My name is Anagha, I am an applications and product development scientist at New England Biolabs. My work focuses on developing nucleic acid extraction kits designed for a wide range of sample types with an emphasis on robustness, scalability and sustainability. And I also work on advancing sample to result workflows for different nucleic acid detection applications.
Jack Martz:
And I'm Jack Martz, I'm one of the technical support scientists here at NEB, I answer customer inquiries about our products full-time. I support a few portfolios including our cloning and DNA assembly technologies and I also support our Monarch® and our protein expression portfolios.
Lydia Morrison:
Excellent. I'm so glad you could both be here with us today. What are the different formats of RNA extraction methods that are available to scientists when they're looking to isolate RNA?
Jack Martz:
Well, historically speaking, organic extractions like TRIzol using phase separation have been the standard method of nucleic acid extraction for several decades. While these methods often have excellent RNA recoveries, the methods have largely become outdated primarily due to the fact that they're labor-intensive and the steps involved to use hazardous materials like phenolic compounds.
Most RNA extractions today rely on solid matrices such as filter-based spin columns, magnetic beads and, under optimal conditions, they bind RNA reversibly on a solid surface. This allows for an easy to use format where the RNA can be washed while it remains bound, impurities get removed that way during the washes and purified RNA can be easily collected or eluded off of the matrix by applying appropriate elution buffer. The choice of what method to use though depends on your user application and what you're wanting to use this for.
Lydia Morrison:
So, what things should people keep in mind, what should scientists keep in mind when they're choosing between column and magnetic bead-based methods?
Anagha Kadam:
So, both silica column and magnetic bead-based methods are widely used and each has its own strengths. The silica column methods are common due to their simplicity and reliability which makes them suitable for most academic lab applications. But there are some drawbacks there, such as limited automation compatibility on open platforms and the significant amount of plastic waste that's generated since each prep uses at least one disposable column. We at NEB try to focus on sustainability in everything we do so, to help reduce environmental impact, our NEB Monarch® nucleic acid purification kits are designed to use considerably less plastic compared to those from other suppliers. It is a small change but it can make a meaningful difference over time.
Also, an alternative approach to silica columns is magnetic bead-based extraction. These methods are also user-friendly but offer additional advantages such as scalability, high throughput processing and compatibility with automation. So, ultimately, the choice comes down to your needs. If you're doing low to medium throughput work and want something simple, columns are great. But if you're scaling up, automating or thinking about efficiency at a larger level, magnetic beads might be the way to go.
Lydia Morrison:
Awesome. I think that breaks it down really clearly. I'm curious, what are some of the challenges that people face when they're working with RNA and are there precautions or recommendations that we make around RNA isolation?
Anagha Kadam:
Yeah, that's a great question. Because RNA is intrinsically unstable molecule and that's something anyone working with RNA learns very quickly, there are a few reasons for that. So, DNA is double-stranded, RNA is a single-stranded molecule and it has chemical features that make it more prone to spontaneous breakdown especially through hydrolysis. But probably the biggest challenge comes from RNases which are enzymes that degrade RNA and the tricky part is that RNases are everywhere. They are in the environment, in biological samples, on lab surfaces and even on our hands and, to make matters worse, they are incredibly stable and difficult to inactivate.
So, when we talk about getting high quality RNA, it really comes down to protecting it at every step from the moment you harvest your samples all the way through storage. And there are some key best practices that can make a big difference. For example, always wear gloves when handling samples and reagents is essential because that helps preventing introduction of RNases from your skin. It's also important to treat your work surfaces and equipment with RNase decontamination solutions, some labs even go a step further and use designated hoods and dedicated areas just for RNA work.
Another important aspect is the chemistry that you're using. RNases tend to be less active under certain conditions so keeping the buffers at a low pH and using chaotropic salts can definitely help protect against RNases. You also want to avoid alkaline conditions because those can promote hydrolysis as well. We also strongly recommend using designated RNase-free reagents and consumables in RNA extraction workflows. If RNase-free plasticware is not available, sterile disposable plastics are generally a good alternative since they are also typically considered to be free of RNase contamination.
So, overall, working with RNA is really about being careful and consistent. Good technique, clean working conditions and the right chemistry all come together to keep your RNA intact.
Lydia Morrison:
So, since RNases can begin even at the beginning of a sample harvest or collection or they can be introduced at any number of steps, do you have recommendations for sample collection and storage?
Jack Martz:
Absolutely. Sample collection practices are one of the most important factors that influence the output of an RNA extraction. In addition to taking precaution for handling and wearing appropriate PPE which my colleague mentioned previously, the use of an RNA stabilization reagent is strongly recommended, this can be commercially available reagents such as RNAlater or StabiLyse™ from Monarch. As samples are collected, users should work quickly to process and break them apart if needed and immediately place them in a tube containing a stabilization reagent and then flash freeze in liquid nitrogen.
If a stabilization reagent is not used, the sample should be flash frozen immediately. All tools and supplies used in sample collection should be RNase-free or sterile and disposable. RNases start acting immediately and any delays in RNA stabilization can cause loss of your RNA integrity and quality as well as skew the biological signal in transcriptomic studies.
After extraction, RNA should be stored at least minus 20C for the short term and minus 80C in the long term. That said, it's best to maintain good lab hygiene when working with your samples after you extract them to avoid contamination in the first place.
Lydia Morrison:
Awesome, thanks. Those are really great things to keep in mind, I think, in order to prevent contamination of RNases. Is there anything else in the RNA extraction process that, if you don't get it quite right, it could lead to misleading data interpretation?
Anagha Kadam:
Yes, there is. In RNA extraction workflows, DNA is a contaminant that can heavily skew end results leading to false interpretations of data especially in transcriptomic analyses. So, for example, both RNA and DNA have the same absorbent spectra which means that, any spectrophotometric quantification, contaminating DNA will also lead to the same measurements leading to overestimation of RNA extraction yields which would be inaccurate. Further, if DNA is present in RNA eluates that are subsequently used for, say, RT-qPCR or sequencing library prep applications, DNA will also get amplified at the PCR steps which will lead to false detection and signal interpretation.
So, it's very important that the DNA be completely removed from RNA before molecular assays are started. To that end, I'd say it's very important to consider an RNA extraction kit that provides DNA removal reagents and enzymes along with the kit to avoid purchasing additional reagents. And a DNA step during the extraction process itself is preferred instead of a post-extraction DNA strip because that can lead to a significant loss of yield. So, really important to take DNA removal steps in RNA extraction.
Lydia Morrison:
Yeah. And using ones that are included in a kit seem like they really take the thought out of that process and eliminate that variable. From a workflow perspective, I'm curious, what's the most critical step in RNA extraction?
Jack Martz:
Most likely cell lysis. So, the fundamental principle of RNA extraction revolves around cell lysis so you have to break open the cell to release the RNA out. Depending on the type of sample, the lysis step can be a simple addition of detergent or chaotropic reagent to disrupt the cell membrane or it can be much more involved for tough-to-lyse samples that are fibrous in nature or contain cell walls such as gram-positive bacteria and plant cells.
Before starting the extraction, users should have an idea of the biological properties of the sample to make the necessary lysis choices. In some cases, lysis reagent-based lysis needs to be supplemented with a lytic enzyme or by using mechanical lysis like bead beading for disruption and sample homogenization. For mechanical lysis, beads are available in different materials, sizes, shapes and the choice of all of those factors is determined by the properties of the tissue including protein content and toughness.
For any unusual sample types, we recommend connecting with us on technical support first that we can provide the most updated technical guidance to help you with your experiment. The lysis steps needs to be optimal to ensure a high yield of RNA.
Lydia Morrison:
So, how do you measure the RNA concentration and what type of quality controls do you use after purification?
Jack Martz:
There are quite a few ways to analyze extracted RNA after purification. By far, the easiest method to measure RNA concentration is by using a microvolume spectrophotometer like a NanoDrop™. Fluorometric assays like the Qubit can be used for low concentration samples and we recommend it for such. And very, very dilute samples sometimes even RT-qPCR is used. The NanoDrop can be used to check for some quality measures as well such as the 260/280 ratio or 260/230 ratio which help detect contaminants such as protein for 260/280 or ethanol, guanidinium, phenol for 260/230. Unfortunately, fluorometric assays or PCR-based assays aren't able to detect contaminants like a spectrophotometer can.
The size of RNA is also an important quality measure. Many labs run their RNA out on a gel or an automated electrophoresis machine like a TapeStation® and the latter can give a quality score based on the sizes of RNA present in a particular sample. This is called a RIN or an RNA integrity number, it's based on the integrity of the ribosomal RNA fragments in the sample. A RIN score is given from one to 10, the higher, the better. Some samples by nature have atypical ribosomal fragment sizes though like in insects so RIN scores would not be accurate in those samples but visual integrity can also be assessed on a gel.
Lydia Morrison:
So, we've talked through a lot of the benefits and comparisons between column-based and magnetic-based, are there other additional factors to consider when choosing an RNA extraction method?
Jack Martz:
Sure. The two that come to mind quickly are elution volume and input amounts. So, the elution volume of a kit can be an important factor to consider when choosing which kit you're going to go with. Many enzymatic steps in a workflow benefit from highly concentrated RNA so having lower elusion volumes can be very helpful. Many kits also have detailed input guidance on how much of your sample you should lyse and load onto the column or the magnetic bead, the total input onto a column is often key for success and overloading or underloading it can lead to undesirable outcomes.
Underloading the column can reduce binding efficiencies so you may see a lower recovery than expected. Overloading it can clog it which, not only can reduce recoveries, but also increase the chance for contamination in your final RNA or have buffer carry over. For mag beads, there's less of an issue with underloading just because those volumes tend to be more scalable.
Lydia Morrison:
Thanks. That's all really helpful things to consider, I think, when making a decision about what kit's right for you and what method you want to go with when you're doing an RNA extraction. I'm curious, if you're new to RNA extraction and you're looking to maybe perform your first RNA isolation or you are working on a project where you need to do an RNA extraction, do you have some advice for someone who's more new to the methodology?
Anagha Kadam:
Yeah. I'd say start with being clear about your goal. If you know exactly what you're trying to get out of your RNA extraction, it makes the process choices easier and you're way more likely to end with high-quality RNA. One of the first things to think about is your sample type. As Jack mentioned, different samples behave very differently, some are easy to lyse, others are pretty tough so understanding those biological properties upfront really helps you decide what kind of lysis approach you will need.
There are several RNA extraction kit choices available on the market, it is definitely worth taking the time to dig into product pages, look at input recommendations, compare performance data and look at what kinds of downstream applications they've been validated for, this can really help you find the best kit for your needs. As an example, if you're trying to get true total RNA including small RNAs like mitral RNAs, you have to make sure the kit is actually designed to capture those. Not all kits do that very well so it's an easy thing to overlook. Another big one is DNA contamination. So, if you're planning downstream molecular applications where DNA could interfere, you'll want a kit with strong DNA removal strategy, ideally something that combines a DNA removal column with a DNase treatment, that's usually a good sign that you'll get cleaner RNA.
And from a practical standpoint, it can be really valuable to choose a more versatile kit, something that works across multiple sample types. This will not only give you more flexibility but can also be a bit more sustainable since you're not buying a lot of different kits from different suppliers. And, finally, don't forget to ask for help. At NEB, we have technical support teams that are made up of scientists who are here to help you design your experiment or troubleshoot as needed, it is a resource for you and it can save you a ton of time.
Lydia Morrison:
Absolutely, scientists just like Jack. Thank you so much, both of you, for being here today, I think you've shared some really helpful information around choosing a method or a kit for RNA isolation or extraction and it's really great to hear, I think, some of the frequent pain points or challenges. And I know that these troubleshooting methods that you've shared and things to consider that you've shared here today are really based on customer questions that we've gotten in the past that our tech support team has fielded and, yeah, just another plug for the tech support team, they're always here to help and I think it's one of the things that makes New England Biolabs really special having access to scientists like you both who are able to help customers work through questions when they might not have a postdoc or another team member at the lab who has time to help them so it's good to know that you're here to help.
Jack Martz:
We're happy to do it.
Anagha Kadam:
Thank you for having us.
Lydia Morrison:
Thank you for joining us for this episode of the Lessons from Lab & Life podcast. We invite you to check out the episode's transcript on neb.com for lots of helpful links from today's discussion around RNA isolation. And we hope you'll be with us next episode which features an interview with microbiologist and virologist Paul Turner from Yale School of Medicine. He joins us to explain how viruses can be used to treat and kill bacterial infections so be sure to come back and catch that upcoming episode.
