The Challenges Related to RNA Extraction for Genome Sequencing

The critical first step to generate a genome after the cell culturing process is to extract the RNA from the mixture. The purpose of this is to break down and isolate RNA from any other cellular components and impurities that are within the culture supernatant. It is important to understand that through this process, they are not separating whole “viral” particles from everything else. They are breaking down the RNA within the sample in order to establish a DNA library for genome sequencing. Many get confused when reading virology papers as they believe that when researchers state that they purified RNA, that this means that the “virus” was properly purified and isolated. Do not get confused with the use of the word purification as they are only speaking in terms of purifying free-floating RNA from various sources, not whole “viral” particles. The RNA is a mixture of many sources, not just the assumed “viral” one. Even then, this “purified” RNA can be in a degraded form and/or full of contaminants. Let’s see if we can break this orocess down a bit and make it a little less confusing:

The Basics: RNA Isolation

“Obtaining high-quality RNA is the first, and often the most critical, step in performing many molecular techniques such as reverse transcription real-time PCR (RT-qPCR), transcriptome analysis using next-generation sequencing, array analysis, digital PCR, northern analysis, and cDNA library construction. To generate the most sensitive and biologically relevant results, the RNA isolation procedure must include some important steps before, during, and after the actual RNA purification.”

https://www.thermofisher.com/us/en/home/references/ambion-tech-support/rna-isolation/general-articles/the-basics-rna-isolation.html

The extraction of RNA is done through various methods and it must be done quickly and carefully as RNA is not as stable as DNA and can degrade rather easily. The 3 main methods used include organic extraction, spin column extraction, and magnetic particle extraction. These methods are outlined below along with their pros and cons in this 2015 article from Roche Life Science:

The top pros and cons of different RNA extraction methods

“Isolating high-quality RNA is the most critical step for successfully performing a broad range of assays, from RT-qPCR or microarray analysis to cDNA library preparation, as well as Northern blot studies. It is even critical for high-throughput transcriptome analysis using next-generation sequencing techniques.

Therefore, getting the most from your RNA isolation procedure is a must. High-quality experiments require high-quality samples, and maximizing yield of non-degraded RNA isolation is key. In this article, we will discuss three of the most common RNA extraction techniques and go over the pros and cons for each strategy.

The organic extraction method

Organic extraction of nucleic acids is historically the most common, tried-and-true method for RNA isolation and removing cellular proteins. This technique requires homogenization of your sample in a phenol-containing solution (usually phenol-chloroform). The phenol-chloroform mixture is immiscible with water, therefore when centrifuged, the samples form two distinct phases.

The lower (organic) phase and phase interface contain denatured proteins, while the less-dense upper (aqueous) phase contains nucleic acids. Importantly, the phase extraction of DNA and RNA is pH-dependent, when the pH is greater than 7.0, both RNA and DNA will resolve in the aqueous phase. A pH less than 7.0, DNA more readily denatures and precipitates into the organic phase and phase interface, with RNA remaining in the aqueous phase.

The aqueous phase containing your RNA is then carefully removed by pipetting (with care not to touch the interface or organic phase, as this can contaminate your sample) and RNA is then precipitated with alcohol and rehydrated for further analysis.

The pros:

• Organic extraction is the gold standard.
• Protocols are well-established and routinely used, making the procedure straightforward for novice researchers.
• Proteins are rapidly denatured and RNA is quickly stabilized.
• The process is applicable to larger samples (such as human or animal tissues) as well as smaller samples from cell culture based experiments.

The cons:

• Not very amenable to high-throughput processing and difficult to automate.
• Manual processing of samples can be laborious.
• Use of hazardous chemical and chlorinated organic waste must be managed carefully.

The spin column extraction method

This is a solid phase extraction technique to bind and isolate RNA within filter-based spin columns. These spin columns utilize membranes that contain silica or glass fiber to bind nucleic acids. Samples are lysed in a buffered solution containing RNase inhibitors and a high concentration of chaotropic salt. The lysates are passed through the silica membrane using centrifugal force, with the RNA binding to the silica gel at the appropriate pH.

The membrane containing residual proteins and salt is then washed to remove impurities, with flow-through discarded. RNA is subsequently eluted with RNase-free water, as RNA is stable at a slightly acidic environment.

The pros:

• Simple, straightforward procedure to perform.
• A ready to use kit format, which adds convenience.
• Amenable to large-scale and high-throughput processing, including automated methods.
• Flexible for use with both centrifugation or vacuum based systems.

The cons:

• Starting with too much sample or incomplete homogenization can clog the membrane and/or result in contamination with proteins or genomic DNA.
• Incomplete cellular lysis can lead to low yields.
• Automation systems for centrifugation or vacuum can be expensive and complex to set up.

Magnetic particle extraction method

This strategy for bioseparation utilizes beads with a paramagnetic core (in other words, they have properties of magnetism only when in proximity to an external magnetic field) coated with, most commonly, a matrix of silica for binding nucleic acids. In this method, cells are lysed in a buffer with RNase inhibitors and then incubated with the magnetic beads, allowing the particles to bind RNA molecules.

The magnetic beads can then be quickly collected by being placed in proximity to an external magnetic field. The supernatant is removed and then subsequently washed and resuspended with removal of the magnetic field. This process can be easily repeated for multiple washes. The RNA is eluted from the magnetic beads with RNase-free water into solution, and the supernatant (containing the pure RNA) can then be transferred.

The pros:

• RNA isolation technique is most amenable to automation and high-throughput methods.
• The magnetic collection and resuspension steps are rapid and simple to perform.
• Rapid and simple magnetic collection and resuspension steps.
• Non-filter method reduces concern for clogging.
• No organic solvent hazardous waste.

The cons:

• Viscous samples can impede migration of magnetic beads.
• While more easily amenable to automation, this technique can be laborious when performed manually with large numbers of samples.
• Risk of contamination of RNA samples with residual magnetic beads.

https://web.archive.org/web/20210226155941/https://www.lifescience.roche.com/en_us/blog/lab-life/dna-and-rna-purifation/the-top-pros-and-cons-of-different-rna-extraction-methods.html

As can be seen, each method has its own advantages as well as limitations and drawbacks. Minimizing the degradation of RNA is the key in order to get accurate results. However, each of these three methods share the risk of contamination. If the RNA is contaminated, downstream analyses such as gene expression studies and PCR amplification can be easily compromised. According to Methods of Enzymology, “no RNA extraction method can completely remove DNA contamination from RNA preparations.” Thus, it could easily be concluded that contamination is bound to occur durimg RNA extraction which requires more treatment (such as with DNase) in an attempt to remove any contaminants. This process of “cleaning” the RNA free of DNA leads to further degradation of the RNA which will ultimately lead to less accurate results. These drawbacks are further explored in the following article:

How to Purify High-Quality RNA

The phenol/chloroform method

“This is the oldest, tried-and-true method for extracting RNA. It uses a straightforward yet laborious protocol that relies on the differing solubilities of molecular species in organic solvents and water. The phenol/chloroform method can accommodate larger samples, but it is low throughout and not easily automated. Incubation times and precipitation temperatures are important, says Matteo Beretta, molecular biologist at PCR Biosystems, who uses low temperatures to protect the RNA from degradation by RNAses. “Attention should be paid not to disturb the phases formed during the process [to prevent contamination of RNA with DNA or proteins], so a good handling ability is required,” says Beretta. “The phenol/chloroform method usually yields a slightly cleaner RNA and allows the extraction of RNA from a lower amount of cells [compared to the spin column method below].”

The spin column/column chromatography method

The spin column method uses small centrifuge tubes that contain silica-based filter columns. Lysed samples treated with RNase inhibitors and high salt concentrations are passed through the spin columns during centrifugation, while the RNA remains bound to the silica within the column. Washing removes impurities, then the purified RNA is eluted from the silica filter using water.

Spin columns, widely available in a convenient kit format, typically use a simple, quick protocol. But this can be complicated by incomplete sample lysis or homogenization, or overloaded filters. “It is important to use an appropriate amount of input material since using too much sample may reduce lysis efficiency, introduce excessive amounts of cellular components other than RNA, and compromise RNA binding to the RNA purification column,” says Danielle Freedman, senior product marketing manager at NEB.”

The magnetic beads method

“This method uses magnetic beads coated with silica or other ligands that bind RNA, such as oligo(dT) to isolate mRNA molecules with intact polyA tails. The beads are incubated with cell lysate and RNase inhibitors, then anchored in place using a magnetic field while the supernatant (containing unwanted debris and impurities) is removed, and the beads are washed to remove lingering impurities. Resuspending the beads in water elutes the purified RNA.”

Common pitfalls and concerns

Inhibiting RNases

The first concern in any RNA workflow is to guard against degradation by RNases. “Work in an environment which is as RNase-free as possible, so wash surfaces and pipettes, use RNase-free (DEPC-treated) water, and change gloves a lot,” says Beretta. Also, add the power of RNase inhibitors as needed. “RNase control is key, so the use of inhibitors specifically, or knowing which conditions cause inactivation of RNases, such as lysis buffers or transport media, together with use of RNase-free consumables, helps maintain [RNA] integrity,” says Andrew Gane, genomics and diagnostics solutions strategy and technology manager at Cytiva.

Sample lysis

The sample type will dictate the appropriate lysis stringency, which can vary widely. This may require optimization, as insufficient lysis means an incomplete yield, while overly stringent lysis can degrade RNA molecules. “The lysis efficiency can be fine-tuned by combining chemical lysis with enzymatic lysis and physical lysis via heat and/or mechanical disruption,” says Markus Sprenger-Haussels, VP, head of sample technologies product development life sciences at QIAGEN. “These parameters have to be well balanced to avoid negative impact on RNA integrity.”

Elution

Elution conditions should be optimized to find the best elution buffer for long-term RNA stability, and also to avoid interference with subsequent downstream applications. For example, azide can affect quantification by spectrophotometry, EDTA can impact PCR efficiency, pH can affect enzymatic reactions, and “addition of carrier RNA might impact [spectrophotometric] quantification or oligo(dT)-primed downstream reactions,” says Sprenger-Haussels.

Contamination with gDNA

Removal of residual genomic DNA (gDNA) from RNA preparations is also an important consideration for some downstream applications, and optimizing workflows can help to reduce gDNA contamination. “Genomic DNA may be carried over from the interphase of organic extractions, or when solid-phase RNA purification methods are overloaded,” says Freedman. “To remove traces of genomic DNA from RNA preparations, samples should be treated with DNase I.”

https://www.biocompare.com/Editorial-Articles/572190-How-to-Purify-High-Quality-RNA/

There are obviously a few issues which can potentially throw off the RNA extraction process. Disturbing the aqueous phase during organic extraction can lead to contamination while using too much sample material during spin column extraction may reduce lysis efficiency, introduce excessive amounts of cellular components other than RNA, and compromise RNA binding to the RNA purification column. When using magnetic beads, there is a risk of contamination from residual magnetic beads. Other pitfalls include the degradation of RNA, too little lysis leading to too little yield, too much lysis leading to RNA degradation, inappropriate elution conditions leading to RNA instability, and contamination from gDNA requiring DNase treatment for removal which can cause further RNA instability. With so many landmines in the way which can cause the breakdown of RNA, it seems somewhat of a miracle there is such a thing as “accurate” results. Any of these alone would be enough to undermine the remaining sequencing processes leading to a contaminated genome.

In order to paint a better picture, presented below are two more sources which look at the various ways in which this process can potentially go wrong. The first is from a study in 2014, it is shown that the DNase treatment used to remove any contaminants during RNA extraction degraded the RNA and diminished yield up to 50%. It is pointed out that variables such as the choice of RNA isolation kits as well as the speed and temperature during centrifugation can significantly impact the amount and quality of the RNA obtained. They concluded that the biggest challenge in RNA isolation is eliminating bacterial DNA contamination which will effect gene expression studies:

Pitfalls of RNA isolation from sputum in COPD

Results: Sputum cell counts varied between 0.95-7´10⁶ cells/g sputum. Isolated RNA content was 332.9±322.2 ng/g sputum. All three processing methods yielded similar amount of RNA, with a slight advantage for DTT (p=NS). Cell integrity was only preserved in DTT. DNase treatment diminished the yield by 30-50%. Surprisingly, in most samples several rounds of DNase treatment was needed to clean the samples from the contaminating DNA. The choice of RNA isolation kit, the speed and the temperature of centrifugation significantly influenced the amount of RNA. The choice of reverse transcriptase had a lesser effect on the PCR products.

Conclusion: The quality and the quantity of sputum RNA depends on several factors during the isolation. However, the biggest challenge is the elimination of bacterial DNA, which is of high importance, since contaminating bacterial background might mask the human RNA in gene expression studies.

https://erj.ersjournals.com/content/44/Suppl_58/P995

Also in 2014, an article came out discussing ways to troubleshoot RNA isolation problems. In it, they highlight important problems such as DNA contamination in RNA, the difficulty in pinpointing the degradation of the RNA, inhibitors in the RNA due to organic salt carryover resulting in protein contamination, and a low yeild of RNA due to mistakes in weighing and/or cell counting.

Troubleshooting RNA Isolation

“As widely used as it is, isolating RNA remains one of the more finicky protocols. Just about anyone who has performed the technique has their own personal tips and tricks to successfully isolate intact RNA from their samples with consistency. Although RNA can be somewhat unpredictable since it is so labile, there are a few common problems that occur that can be solved.

1. Problem: Genomic DNA in the RNA

The RNA elutes with genomic DNA as evidenced by high molecular weight smearing, or it appears clean on a gel but -RT controls amplify when PCR is performed.

Cause: No matter what method you use for RNA isolation, traces of DNA always carry through. This is true with TRIzol (phenol) preps and with silica spin filters. This can be caused by insufficient shearing of the genomic DNA during homogenization. If using phenol method, the pH of the phenol is key (it should be acidic) and your skill in pipetting only the aqueous phase will result in more or less DNA contamination.”

2. Problem: Degraded RNA/ low integrity

“The rRNA bands appear smeared on a gel or the 18s is more intense than the 28s band. On the Agilent Bioanalyzer, you see a bigger 18s peak.

Cause: Degradation occurred at some point during processing. This can be difficult to pinpoint. It could have happened during collection and storage, or possibly during extraction. It could also have occurred post-isolation.”

3. Problem: Inhibitors in the RNA

“The RNA has an abnormally low 260/230 reading (below 1.0) or 260/280 reading or does not work in reverse transcription.

Cause: A low 260/230 in an RNA prep is indicative of guanidine salt carry over into the sample or organic inhibitors (such as humic acids or polysaccharides if the sample is environmental). Guanidine salts are used in TRIzol and in silica preps. These salts inactivate RNases, but will also inhibit proteins such as RT enzymes if present in the final RNA.  A low 260/280 measurement indicates protein contamination.

4. Problem: Low yields of RNA

“The yield of RNA is lower than expected- either based on your previous results, or, based on reported yields for a certain tissue or cell type.  RNA yields can vary greatly between different cultured cell types and in different tissues. For blood RNA, it can vary from person to person.

Cause: If the yield of RNA is lower than you expected or know it should be, and the RNA is intact (read: not degraded) , then the homogenization may not have been complete. To isolate RNA, a strong lysis is key. Tissues stored in RNALater will tend to be a little more difficult to homogenize. Low yields could be caused by mistakes in weighing of tissue or in the cell counts for cultured cells. You may have less cells than you think. With blood RNA, the buffy coat can vary based on your skill in collecting the white cell layer and each individual patient.”

Troubleshooting RNA Isolation

In Summary:

• Obtaining high-quality RNA is the first, and often the most critical, step in performing many molecular techniques such as reverse transcription real-time PCR (RT-qPCR), transcriptome analysis using next-generation sequencing, array analysis, digital PCR, northern analysis, and cDNA library construction
• High-quality experiments require high-quality samples, and maximizing yield of non-degraded RNA isolation is key
• The 3 main methods of RNA extraction are:
  1. Organic extraction
  2. Spin column extraction
  3. Magnetic particle extraction
• Each method has its own drawbacks and limitations
• With the organic extraction method, the aqueous phase containing RNA needs to be carefully removed by pipetting with care not to touch the interface or organic phase, as this can contaminate your sample
• With the spin column extraction method, starting with too much sample or incomplete homogenization can clog the membrane and/or result in contamination with proteins or genomic DNA
• With the magnetic particle extraction method, there is a risk of contamination of RNA samples with residual magnetic beads
• Other issues include:
  1. Inhibiting RNase leading to the degradation of RNA
  2. Insufficient lysis leading to an incomplete yield or overly stringent lysis degrading RNA molecules
  3. Improper elution conditions leading to long-term RNA instability and interference with subsequent downstream applications
  4. Carryover of contaminating genomic DNA from the interphase of organic extractions, or when solid-phase RNA purification methods are overloaded
• Regarding the extraction of RNA from sputum, one study found that eliminating bacterial DNA contamination was the biggest challenge which can ultimately mask human RNA
• DNase treatment diminished the yield by 30-50% and in most samples several rounds of DNase treatment was needed to clean the samples from the contaminating DNA
• The choice of RNA isolation kit, the speed and the temperature of centrifugation significantly influenced the amount of RNA
• No matter what method used for RNA isolation, traces of DNA always carry through
• Skill in pipetting only the aqueous phase will result in more or less DNA contamination
• Degraded RNA/ low integrity is difficult to pinpoint and can occur during collection and storage, during extraction, or it can occur post-isolation
• A low 260/230 in an RNA prep is indicative of guanidine salt carry over into the sample or organic inhibitors and indicates protein contamination
• RNA yields can vary greatly between different cultured cell types and in different tissues
• Low yields could be caused by mistakes in weighing of tissue or in the cell counts for cultured cells

RNA purification is not the same as the initial steps that are supposed to be used when purifying and isolating “viruses” in order to prove their existence and pathogenicity. As defined by Science Direct, total RNA isolation is the method that helps to separate pure RNA from tissues and the mixtures of DNA or proteins. Essentially they are breaking down everything in the sample chemically into RNA so that it can be used for various genomic applications. Whole “virus” particles are not obtained in this process. The resulting “pure” RNA is a mixture from many sources. However, it is clear that the methods used for the extraction of RNA either from cell culture or straight from clinical samples are fraught with potential problems regarding contamination and RNA degradation. Any errors in this first crucial step will affect the following steps in the sequencing process and lead to problems with the reliability and accuracy of sequencing a genome. This means that the puzzle pieces used to create the picture will be inaccurate. If the pieces of the puzzle are incorrect, what does that say about the puzzle?