RNA purification - Opinions & Insights
The Science Advisory Board surveyed life scientists on their experience with RNA purification; specifically, the use of DNA microarrays and/or real-time PCR and the factors they take into consideration when preparing the starting material for these applications.
When asked to provide a comment to the following question, " What is the greatest challenge that you face with regards to RNA purification?," The study's
participants responded with the comments that appear below.
Each response has been edited for grammar and clarity and the respondent's first name, scientific specialty, job position, and geographic region appear next to each comment.
Ability to isolate sufficient quantities of small RNAs from a small number of cells that represent microRNA pools and use these to construct good representative libraries.
Raju, Staff Scientist, North America
Being able to purify RNA from single cells with acceptable quality.
Tiziano, Staff Scientist, North America
Clean working procedures.
Urs, Post Doctoral Fellow, North America
Concern that the RNA species profile present in an extract reflects intracellular one (i.e., degradation, purification process may affect some RNA species more than others).
Herv, Principal Investigator, Europe
Confidence that sample is intact and remains so during/after extraction.
Clint, Professor/Teacher, North America
Consistently high yields, especially from small starting numbers of cells, such as from clinical samples.
Jeff, Principal Investigator, North America
Contamination/RNase.
Simon, Professor/Teacher, North America
Contaminations.
Gabriele, Post Doctoral Fellow, North America
Convenience, optimized to my protocol, price and time-to-results.
Rosenda, Staff Scientist, Central/South America
Degradation and low yield.
Wenjin, Graduate Student/Research Assistant, North America
Degradation and yield.
Devanjan, Post Doctoral Fellow, North America
Degradation of RNA.
Shyam, Professor/Teacher, North America
Degradation of RNA.
Xiang, Staff Scientist, North America
Degradation of RNA.
Kuang-Ren, Principal Investigator, North America
Degradation of RNA.
Wendy, Graduate Student/Research Assistant, North America
Degradation of RNA.
Kuan-Teh, Principal Investigator, North America
Degradation of RNA.
Tatyana, Post Doctoral Fellow, Europe
Degradation of RNA.
Tzong-Jen, Post Doctoral Fellow, North America
Degradation of RNA.
Namita, Staff Scientist, North America
Degradation of RNA.
Nicola, Graduate Student/Research Assistant, Africa
Degradation of RNA.
Sheila, Graduate Student/Research Assistant, Central/South America
DNA contamination in basic prep - so we digest with DNaseI, then further purify sample via a Qiagen RNeasy kit.
Grant, Staff Scientist, Australasia/Pacific
DNA contamination.
Shu, Graduate Student/Research Assistant, North America
Early stage conservation of RNA (freezing???) in the tissue.
Markus, Laboratory Technician, Europe
Ease, purity, adaptability to subsequent steps, ability to purify RNA at lower and lower limits of detection, i.e., smaller quantities. What needs to be done is to produce machines that can produce analysis of those tiny quantities of RNA and also to develop methods to manipulate those tiny quantities of mRNA, and other specific RNA purifications and fractionations at those low levels.
Paul, Principal Investigator, North America
Efficient removal of polysaccharides and polyphenols from plant tissue.
Andrew, Post Doctoral Fellow, Australasia/Pacific
Elimination of RNase activity and stability of message (especially longer messages).
Nicholas, Lab Director/Supervisor/Coordinator, North America
FFPE is the challenge. The rest is easy.
Stephen, Staff Scientist, North America
For total RNA purification, RNA degradation is the greatest challenge that I have faced. As for miRNA purification, to get sufficient and clean miRNA sample is a problem. Moreover, the miRNA purification process is time and labor intensive.
Sook Peng, Graduate Student/Research Assistant, Asia
Getting adequate yield and quality from the very small amounts of material in archived stereotactic biopsy samples.
James, Post Doctoral Fellow, Europe
Getting high quality RNA from difficult tissues.
Penny, Principal Investigator, North America
Getting high yield from primary cells and maintaining high quality RNA.
Mark, Principal Investigator, North America
Getting the best yield for each sample. I work with varying sources of RNA such as Xenopus embryos and field lichens so the sample preparation can be difficult.
Charles, Professor/Teacher, North America
Getting uniform and consistent amounts of RNA from formalin fixed sections.
Arun, Lab Director/Supervisor/Coordinator, Europe
Greatest challenge is scaling up with minimal cost, based on kits which add convenience, but cost.
Donna, Professor/Teacher, North America
High enough yield from lower numbers of cells.
Richard, Graduate Student/Research Assistant, North America
High yield and ultra pure! For polyA mRNA without ribosomal RNA.
Alex, Principal Investigator, Europe
High yield, yet high quality (no DNA contamination).
Eric, Post Doctoral Fellow, North America
How to obtain material with high yield, good quality and minimal hands on time.
Ivan, Graduate Student/Research Assistant, North America
I find the Qiagen kit to be a very easy to use kit that produces high quality results. However, more often than not I have very little recovery and many times no recovery. I think the silica gel column retains a lot of the RNA, and it is difficult to elute. If you really need a high quality sample this is a good kit to use however if you have precious little sample to work with I would not risk it on a Qiagen column. Traditional GITC/phenol/chloroform methods work much better.
Kimberly, Graduate Student/Research Assistant, North America
I haven't seen any good method to remove DNA contamination.
Dohun, Post Doctoral Fellow, North America
I think that a superb RNA isolation kit should focus on three things: speed of preparation, protection from degradation during the whole procedure and low price.
Juan, Principal Investigator, Central/South America
I think the biggest challenge is eliminating DNA contamination and getting a good yield from a limited amount of tissue is also important. Finding a good method of homogenizing, particularly skin samples is also important because grinding skin in liquid nitrogen is a long process.
Tom, Laboratory Technician, North America
I usually extract RNA from animal or human brain tissue. The use of kits like RNeasy (Qiagen) tends to give low yield of RNA due to the presence of brain lipids. Therefore, I use a two-step procedure. First, I purify the RNA with homebrew GdCN-reagent or TRIzol and in a second step apply this "crude" RNA to the RNeasy spin columns to obtain pure RNA with high yields.
Ludwig, Staff Scientist, Europe
I will like to see two kinds of products on the market: 1. A modification of TRIzol/RNAStat that can also take better care of DNA contamination. 2. Some spin column system that can be used to isolate mRNA from total RNA at some reasonable price. The advantage will be that we can start with a smaller volume and the technique will be faster than gravity flow.
Alfica, Post Doctoral Fellow, North America
In a clinical setting, with a constant turnaround of relatively inexperienced staff, training will always be an issue. It is much more straightforward to teach someone how to use a kit, than have them work from scratch.
David, Lab Director/Supervisor/Coordinator, Europe
In bacterial samples to completely remove genomic DNA.
Ivana, Post Doctoral Fellow, Europe
Isolating RNA from limiting amounts of human tissue, such as small tumor biopsies, is difficult, and a method for improving recovery would be very welcome. Perhaps the inclusion of a carrier that was neutral in downstream reactions and provides a scaffold specifically for RNA could be developed?
Oliver, Principal Investigator, North America
It is most difficult thing to purify from tissue. Tissue from prostate or something like have many enzyme.
Megumi, Staff Scientist, Asia
It's very easy now to get high quality RNA in good yield using available kits. The biggest challenge for me with regards to RNA purification is turn around time. It still takes the better part of a day to purify RNA from samples.
Lars, Graduate Student/Research Assistant, Europe
Keeping all RNase free.
Heather, Laboratory Technician, North America
Keeping it pure and in good condition.
Jani, Professor/Teacher, North America
Keeping reagents stable and avoiding cross-contamination along with avoiding contamination with RNases are the greatest challenge with regards to RNA purification.
Persis, Post Doctoral Fellow, North America
Kits are necessary, but not sufficient for intact and pure RNA. I found them too expensive, given that you can manufacture your own.
Claudio, Staff Scientist, Central/South America
Lack of DNA or phenol contamination. It is still hard to eliminate all DNA or phenol in our samples run in a 96 well format.
Alex, Staff Scientist, North America
Lower the cost, increase the yield.
Edgardo, Professor/Teacher, Europe
Lysing cells.
Tanya, Principal Investigator, Europe
Maintaining RNA purity and integrity throughout the isolation procedure when using different sources of material (cell lines, blood, etc.).
J, Principal Investigator, North America
Maximizing yields from highly degraded (e.g., paraffin-embedded) tissue samples.
Tim, Staff Scientist, Europe
mRNA yield.
Farida, Principal Investigator, North America
My environment is relatively conservative when it comes to automatization of RNA purification methods. Any robotic method or instrument must have a proven track record, with associated publications in peer-reviewed journals, in order to receive the blessing of our higher management at the time of purchase. This is probably because the process is parallel to our main expertise, as opposed to central or integrated with it. If robots were cheaper, the side-task of RNA purification could probably take sufficient importance to warrant a purchase. As it stands, because RNA purification and isolation are only tools that serve a higher purpose, any method that gets the work done will do.
Dany, Lab Director/Supervisor/Coordinator, North America
My greatest challenge is obtain RNA from uncommon sources like infected insect vectors, because I have to determine empirically the method I must follow to obtain the best quality RNA and yield that I can.
Jorge, Principal Investigator, Central/South America
No real challenges. Been doing this for about 15 years so it is pretty routine.
Chris, Principal Investigator, North America
No RNA degradation and no DNA contamination. Bead must be the solution to that.
Frank, Lab Director/Supervisor/Coordinator, Europe
Not having to rework protocol every time for a different cell line or more importantly, when looking at cells that have been treated (with growth factors, stress pathway activators, etc.).
Ri-Chee, Staff Scientist, North America
Obtaining a good yield with sufficient purity using a small amount of tissue.
Suchitra, Post Doctoral Fellow, North America
Obtaining good quality RNA for downstream applications.
Annalese, Post Doctoral Fellow, Australasia/Pacific
Optimal percent recovery of starting amount of RNA.
Steffney, Lab Director/Supervisor/Coordinator, North America
Optimizing purity, yield and efficiency.
David, Professor/Teacher, North America
Overcoming RNA degradation.
Permila, Principal Investigator, North America
Precipitation of random RNA pool occurring even with traces of magnesium.
Alexander, Post Doctoral Fellow, North America
Prevent RNA samples from degrading due to RNase contaminations.
Marc, Staff Scientist, Europe
Product yield is the most important challenge, then RNase contamination.
Yavuz, Post Doctoral Fellow, North America
Purification from small samples from laser capture micro-dissection.
John, Professor/Teacher, Europe
Purification is easy. Quantification is our biggest problem - often there is contaminating DNA or nucleotides (from in vitro transcription, which happens even when using commercial cartridges) or aurin tricarboxylic acid (a RNase inhibitor). All of these things interfere with getting an accurate assessment of quantity.
Margaret, Principal Investigator, North America
Purifying RNA from formalin-fixed tissues.
Yan, Staff Scientist, North America
Purity, yield and problems with degradation.
Nadia, Professor/Teacher, Europe
Purity.
David, Lab Director/Supervisor/Coordinator, North America
Quality and amount of RNA, dynamic range of various RNA species and stability.
Ramars, Post Doctoral Fellow, North America
Quality and integrity of RNA and avoiding DNA contamination.
Cornelius, Purchasing Agent/Buyer, North America
Quality and quantity from small sample sizes.
Austin, Principal Investigator, North America
Quality and stability are always an issue with RNA.
Jamie, Principal Investigator, North America
Quality and stability.
Sarabjit, Principal Investigator, Europe
Quality of RNA and ability to scale down for small samples.
Jeff, Principal Investigator, North America
Quality versus time is a trade-off.
David, Staff Scientist, Australasia/Pacific
Quality, and the percent recovery from column purification. Often we purify small amounts of RNA via columns and the percent recovery can be quite limiting.
Tim, Post Doctoral Fellow, Australasia/Pacific
Reliable automation for higher throughput.
Hinrich, Lab Director/Supervisor/Coordinator, Europe
Removing RNA and phenolics, which are abundant in my plant samples. I prefer commercial kits; the only problem is the amount to be extracted is minimum.
Ahmad, Principal Investigator, Asia
Representation at end of purification that is comparable to the cell in terms of copy number. This then becomes a very easy tool for quantifying expression.
Samiwala, Department Head, Asia
Reproducibility and loss of low abundant species.
Philip, Lab Director/Supervisor/Coordinator, Europe
Reproducibility and stability.
Ralph, Principal Investigator, North America
Reproducibility.
George, Professor/Teacher, North America
Reproducibility.
Bernt, Professor/Teacher, Europe
Reproducibility.
Leslie, Lab Director/Supervisor/Coordinator, North America
Reproducibility.
Patrick, Principal Investigator, Europe
RNA degradation or no DNA when trying to get RNA and DNA from the same sample using different methods.
Julia, Graduate Student/Research Assistant, North America
RNA integrity and quality are obviously issues that can be addressed by innovative commercial kits, but nothing beats a good lab technician!
Michael, Post Doctoral Fellow, Europe
RNA isolation is still is a technique that requires a lot of technical skill. Because of high turn over of technicians, getting them trained again and again in the precautions to avoid contamination and degradation of RNA is the biggest challenge.
Margit, Principal Investigator, North America
RNA purification from 50 cells.
Masoud, Graduate Student/Research Assistant, Asia
RNA purification is a widely used method. It's a foundation for a variety of applications and the quality of this step can't be overestimated. Thus, comparative analysis of different methods is very important. In my opinion, one of the future questionnaires could focus on other issues of RNA isolation like: sensitivity limits or ability to isolate RNA from single cells. I think that RNA isolation will move towards these directions, because the main caveat of RNA isolation today is that RNA comes from a heterogeneous population of cells (in tissue - there are many cell types forming particular tissue, in cell culture - cells are not synchronized in their cell cycle). I would be very interested in knowing opinion of other researchers on that important issue.
Andrzej, Post Doctoral Fellow, North America
RNA quality and lack of DNA contamination.
Adrienne, Principal Investigator, North America
RNA Quality is extremely important for downstream applications such as cDNA synthesis and qPCR. I have evaluated 96 well isolation kits from numerous vendors and find them all to be extremely unsatisfactory All of them give reasonably good yields when a large numbers of cells are used (such as 106). However, as the number of cells decreases and especially if the cells are undergoing apoptosis, it seems that there is no 96 well kit that does a reproducibly satisfactory job of RNA isolation for downstream qPCR applications.
Raghu, Staff Scientist, North America
RNA samples obtained with TRIzol reagent from GibcoBRL were further treated with DNase I of Roche Diagnostics.
Bartolome, Principal Investigator, Europe
RNA stability and DNA contamination.
Christopher, Post Doctoral Fellow, North America
RNA stability upon storage.
Robert, Principal Investigator, Europe
RNase activity - genomic DNA contamination for subsequent RTPCR analysis.
David, Staff Scientist, Europe
RNase being present in equipment.
Fransiscus, Post Doctoral Fellow, North America
RNase contamination and RNA stability.
Jay, Graduate Student/Research Assistant, Asia
RNase contamination.
Andrea, Principal Investigator, North America
RNase contamination.
Elizabeth, Post Doctoral Fellow, North America
RNase.
Richard, Principal Investigator, North America
Scalability and automation, without sacrificing yield and quality, are the biggest problems we face with regard to RNA purification.
Dominick, Staff Scientist, North America
Scalable yield. I need to isolate RNA (total or PolyA) from large samples like rat kidneys and from small samples like cell pellets. So far, I'm having most trouble with the smallest samples.
Laurie, Staff Scientist, North America
Scaling down the sample size to a few milligrams of tissue from patients.
Wolfgang, Lab Director/Supervisor/Coordinator, Europe
Small sample size from specific tissue.
Joseph, Post Doctoral Fellow, North America
Solubilization without shearing after ethanol precipitation.
Wayland, Staff Scientist, North America
Speed, yield and DNA contamination.
Zen, Post Doctoral Fellow, Europe
Suitable quality and quantity for downstream uses.
Tracey, Principal Investigator, Europe
The biggest challenge we face in RNA purification today is throughput. Our progress could be further advanced if a system was available which was capable of purifying RNA, of high quality and consistent yield from small numbers of cells (2000-5000) in a 384-well format.
James, Staff Scientist, North America
The critical step is not to degrade your samples at any stage and to make sure that the extraction yield and quality is reproducible between all your samples from a particular study.
Marc, Principal Investigator, Europe
The future resides in micro- and even nano-scale analyses, e.g., microfluidics, single cell analysis. The challenge is then to be able to isolate, purify, and quantitate RNA at that scale.
Lillian, Lab Director/Supervisor/Coordinator, North America
The greatest advantage of RNA purification kits is their ability to purify RNA within a couple of hours. This is great for preserving RNA integrity. However, the most common downside I noticed is DNA contamination. Inclusion of a DNase digestion step in the protocol invariably results in loss of RNA integrity. An excellent RNA purification kit would be that which has an integrated DNA elimination step, without compromising RNA quality. (Ambion has a kit for this purpose, but it is not very effective.).
Vashisht, Post Doctoral Fellow, North America
The greatest challenge for RNA purification from tissue is throughput.
Jean, Lab Director/Supervisor/Coordinator, North America
The greatest challenge I find is keeping the tissue quality intact. We snap freeze each organ from the rat in liquid nitrogen and store them at -80C until isolation. If not frozen quick enough, we tend to see degradation. To combat this, we grind our tissue into powder in liquid nitrogen and aliquot into ~100mg per tube.
Christopher, Lab Director/Supervisor/Coordinator, North America
The greatest challenge is getting enough RNA from very small sample sizes.
Bob, Staff Scientist, North America
The greatest challenge is probably dealing with the phenol. If a company can make a cost-efficient product that circumvents the use of phenol, then we would seriously consider purchasing from that company.
Joe, Principal Investigator, North America
The greatest challenges of RNA purification remain the same: cost/effectiveness, purity, yield.
Giuseppe, Principal Investigator, North America
The increase in time to extract a sample resulting from scale up is a challenge. Commercial kits are very good for small tissue samples of 100mg or less but when you have to extract RNA from a fruit, which normally contain very low concentrations of RNA as well as lots of interfering polysaccharides, there are no products available that allow the same throughput as there is for smaller starting tissue amounts. For example, we go from being able to easily extract about 10-20 small samples from leaf in half a day to only 2-4 fruit samples (10g starting weight) in 3 days. Naturally, we would like to speed this up.
Sean, Staff Scientist, Australasia/Pacific
The most important and critical limiting factor is the sample quality. Very challenging is the task of getting the best sample quality. I assume that many studies rely on "wrong" data because of degraded RNA.
Frank, Principal Investigator, Europe
The purification of small RNA species (i.e., micro RNAs and siRNAs) from cells and tissues is most challenging with respect to backgrounds of other RNA species and a general background of nucleic acids in the sample.
Jens, Graduate Student/Research Assistant, Europe
The reproducible purification of total RNA from tissue containing large amounts of proteoglycan remains a significant obstacle. Tissues such as cartilage that contain low cell numbers and significant levels of proteoglycan are still difficult to work with using conventional reagents. Diseased tissue that may contain still fewer cells is largely refractory to meaningful RNA analysis.
Matthew, Graduate Student/Research Assistant, Europe
The RNA purification methods, particularly commercial kits, have improved significantly over the past 15 years. The greatest challenge currently is ensuring that biosamples are harvested, handled, and stored properly prior to the RNA purification, particularly if laser capture microdissection is to be used.
Karen, Principal Investigator, North America
The RNA yield and quality seems the biggest contradictory problem we face now.
Xiao-Lu, Staff Scientist, North America
The sample stability.
Esther, Professor/Teacher, Central/South America
The yield and quality is a major issue- I purify RNA from bacteria and fungal isolates but mostly from environmental samples such as soil/sludge. This proves difficult. The use of a glass fiber or silica spin column (kit) after homebrew purification is optimal since the sample size can be large with an initial large yield but poor purity. After the use of a commercial spin column RNA purification kit the purity is >1.6 (260/280).
William, Principal Investigator, Africa
The yield and storage.
Yin, Graduate Student/Research Assistant, North America
Time and effort per RNA quality should approach zero!
Thomas, Lab Director/Supervisor/Coordinator, Europe
Time consuming and very prone to degradation.
Sandra, Graduate Student/Research Assistant, Europe
Tissue disruption and degradation of the RNA.
Tricia, Post Doctoral Fellow, North America
To get an extraction efficiency of 100%, which is critical at the time of detecting/quantifying viruses in the environment.
Veronica, Post Doctoral Fellow, North America
To keep RNA from degradation during purification; to isolate RNA from tissues with high content of RNases (pancreas); to limit degradation during storage.
Anna, Staff Scientist, North America
To minimize the amount of contaminations with genomic DNA.
Stefan, Lab Director/Supervisor/Coordinator, Europe
To prevent degradation of RNA and increase yield.
Amit, Post Doctoral Fellow, Asia
To visualize well the phase separation.
Gillian, Staff Scientist, Africa
Trying to maintain consistency from batch to batch.
Jeremy, Post Doctoral Fellow, North America
Unfortunately RNA separation is not as cheap, quick and easy as I'd like it to be.
Georgina, Principal Investigator, North America
With respect to my work, the greatest challenge is the time interval between isolation of RNA and its electroporation into mammalian cells. So, it is imperative that I get excellent purity and very stable yield for my purified samples, to guarantee the consistency between experiments.
Kalliopi, Post Doctoral Fellow, Europe
Worried about RNase contamination. Need to be aware of everything that gets touched.
Aline, Post Doctoral Fellow, North America
Yield and degradation.
Virginie, Post Doctoral Fellow, North America
You always would gain on every matter: price, time invested on RNA preparations, yield.
Toni, Staff Scientist, Europe
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