RNA Extraction - Science method

Dear Morvarid,

There are a number of methods to extract RNA. If you are intending to use an RNA extraction kit, any reliable company offers high quality kits. However, more conventional methods also result in high quality RNA. Depending on your aim (whether you test blood cells in an experiment or not) RNA can be extracted from blood and from isolated PBMC. In order to have high quality RNA, you must have highly viable cell pellet. Either extract RNA right after spinning your cells, OR snap-freeze using liquid nitrogen and store in a reliable -80 freezer until RNA extraction.

Sabine Strehl alright then i will try doing that thanks again!

the glass surface is likely to adsorb DNA/RNA, it's better to use some other beads such as ceramic or metal https://lab.plygenind.com/mastering-bead-selection-for-effective-homogenization

Hi,

Did you find a way to do the RNA and plasmid DNA extraction from the same samples?

I'd try isopropanol to remove the formamide. Should be more efficient than ethanol (at least from experience with precipitating nucleic acids from aqueous solutions). Basically, any solvent that is partially miscible with formamide should do the job, as long as the RNA doesn't dissolve in it. So, in the end, it will precipitate.

Laia-M. Pardinilla back when i worked in the diagnostic laboratory, i used CyBio Felix from AnalyticJena. It is also based on magnetic beads technology and it is fully automated, you can modify the program, customize configurations.. and we had two of them, so we could process 192 samples at once, i really liked that one https://www.analytik-jena.com/products/liquid-handling-automation/liquid-handling/flexible-benchtop-liquid-handling/cybio-felix-series/

Usama .. thanks for your response. Please I'll like to know if it's possible to do relative quantification without endogenous genes as he earlier mentioned. Thanks

Thanks a lot Laura Leighton ! I really appreciated! You actually mentioned something that I have totally forgot before (about the use of column kit to purify the RNA). In fact I'm trying to avoid it from the beggining, but the principle that the beads could block the column can be applied for other similar/procedures as well... Thanks a lot for your answer and share your thoughts!

For cDNA synthesis, high-quality RNA with an A260/A280 ratio of 1.8 to 2.2 is ideal, and a minimum concentration of 1 μg/µL is usually recommended. For Real-Time PCR, the optimal RNA concentration is highly dependent on the gene of interest, and can range from 1 ng/µL to 500 ng/µL.

Compare the amplification of samples like this to other more pure samples. If they cluster tightly around the same Ct, and the curve looks exponential, then it's most likely OK. I've also done real time for microRNA applications using SYBR green chemistry, was OK for me. You can check for inhibition by making dilution series, 1:10 would be ok especially if there's an abundant microRNA. See if you can find anything that has a Ct of 20-21 ish, with that you can make 1:10 dilutions (5-6 of them) or you could even check with 1:2 (half-half) dilution if your microRNAs amplify late (around after 30-33 Cts roughly). If you do 1:2 dilutions (that is half diluent half cDNA), remember your Cts will differ by roughly 1 (i.e. take 1:20 cDNA, make it 1:40, then 1:80 etc). So in the above case, if 1:20 came out at 25 ct, then the 1:40 should come at 26 Ct if amplification efficiency is near 100%. If you generate a 5-6 dilution series, you can get an idea if there is inhibition. I hope that makes sense to you.

Dear Linn Amanda Syding

Thank you for this great insights. Do you think that we have to check the integrity of RNA isolated by those different ways?

It should be fine:

RNA in solution at room temp is far more stable than people think: the real danger is RNAse activity, which will be effectively zero in trizol (because you will have denatured all the RNAases).

It's not _ideal_, in that -80 storage is absolutely a better idea, but you don't really lose much (beyond a couple of hours of time) just extracting the RNA and having a look.

Alternatively, stick the samples in -80 and run the extraction alongside other samples in future.

Hello Shreyas Gujar

You shouldn’t have forgotten to keep the sample at -80 degree C before leaving the lab. Anyway, such incidences keep happening! For your information, RNA is stable in trizol which deactivates RNases. However, the sample can be kept at room temperature at this point for, at most, a few hours, or the sample can be frozen for later use at -80°C.

So, you should not proceed with RNA extraction for this sample which has been kept at room temperature for two days.

Best.

Are you sure freeze/thaw is the best idea here? One of the main problems with RNA isolation from cells is that as soon as you disrupt the cells, all sorts of RNAses get liberated from their normal cellular context and start just chewing up anything they can find. By repeated cycles of freeze thaw, you're essentially bursting the cells and letting the RNAses do their thing, multiple times.

If I were doing this, I'd removed the supernatant from the wells, wash 1x with PBS, and then literally just dump trizol (or equivalent) straight into the wells and squooge around with a cell scraper. This would be what I use for RNA isolation. Addition of trizol (which contains chaotropic salts and bucketloads of phenol) unfolds everything near enough instantly, and stops RNAses working.

The other issue you have is that 260/230 is a ratio, so a low value can reflect salt contamination (high 230), low [RNA] (low 260) or some combination of the two. And you don't have a lot of RNA (unless you have a large volume at 50ng.ul-1).

You can reprecipitate your RNA, as Mohamed Khashan suggests. Reprecipitation usually brings down 'most' of your RNA but much less of your contaminating salts, and also allows you to decide what an appropriate volume would be for dissolving the pellet (i.e. a tiny pellet could be dissolved in 10ul, while a massive pellet might warrant 50ul).

It is generally not recommended to use a viral RNA extraction kit for the isolation of human RNA, as the kit is specifically designed for the isolation of viral RNA and may not efficiently extract human RNA. Additionally, the reagents and protocols used in the kit may not be optimized for the isolation of human RNA and may lead to poor quality or quantity of RNA. It is recommended to use a kit specifically designed for the isolation of human RNA, such as the Quick-RNA™ MicroPrep Kit-Zymo Research.

RNA cleanup kits, such as the NEB Monarch #T2030S, are typically used to purify and concentrate RNA samples prior to downstream applications such as reverse transcription (RT) and quantitative PCR (qPCR). These kits can help to remove contaminants such as DNA, salts, and proteins that may interfere with downstream reactions.

It is important to note that even after a DNase I treatment, DNA contaminants can still be present in your RNA sample. So, if you are performing downstream applications that are sensitive to DNA contamination, such as RT-qPCR, it is recommended to use a RNA cleanup kit to purify and concentrate your RNA sample.

RNA cleanup kits can help in several ways, including:

-Removing any remaining DNA contaminants after DNase I treatment -Removing salts and other contaminants that may interfere with downstream reactions -Concentrating the RNA sample to improve downstream sensitivity and specificity

In my experience, RNA cleanup kits are an important step in the purification and concentration of RNA samples, and can help to improve the sensitivity and specificity of downstream applications such as RT-qPCR. However, it's worth noting that some RNA purification methods are based on selective binding properties of the RNA molecules to specific silica beads, and this can lead to bias in the recovery of different RNA species. Therefore, it's always good to check the recovery rate of your target RNA species after purification.

To sum up, it is recommended to use an RNA cleanup kit for downstream applications of your RNA sample, particularly if you are performing RT-qPCR, to remove any remaining DNA contaminants, to remove salts and other contaminants, and to concentrate the RNA sample. However, it's worth checking the recovery rate of your target RNA species after purification.

I have the exact same problem using this kit, did you ever find a solution to this? I have also carried out a DNase step but my 260/230 values are still very low!

To deal with possible RNAses, dissolve agarose in TAE/TBE buffer containing 2% Hypochlorite (v/v). That should inhibit RNases.

It shouldn't be a problem but you should move your samples to a -20 refrigerator soon. Samples in Trizol can stay viable upto 6 months at -20.

Yes, you can use keep your homogenised samples in dry ice for 3-4 hours. It is safe and will not affect the result

Dear John Hardy Lockhart,

Appreciate for your suggestion.

As a rule of thumb, 35-36 cycles is enough to amplify just about anything (i.e. a single starting template molecule), and for qPCR you can usually assume a Cq of ~35 represents the absolute lower limit of detection.

Accordingly, Cq values of ~34 = 2 molecules, ~33 = 4, and so on.

A reference gene giving a Cq of 30 means there are countable numbers of reference transcripts in your wells (~30-40 or so), and this should never be the case for a reference (which should generally be fairly abundant). You have very little starting material, and the reference is telling you this.

Remember, you...cannot have less than one molecule in a well. PCR doesn't work on "half a template" or "0.001% of a template": there's a molecule there, or there isn't.

Getting a Cq at cycle 50 means it isn't there.

Alternatively, you could have a ruinously inefficient PCR, in which case you'll see what _should_ appear at cycle 25 actually appear at cycle 50 (or whatever), and if this is the case, your data is meaningless. This isn't so bad, though: you can always redesign primers to get a more efficient PCR reaction.

why my total rna on gel looks like oneband at the top and a smear on the bottom not a clear band..the rat tissue was stored in trizoand then processed in qiagen kit...i am unable to understand why it came like this..please help

All RNA extraction and library preparation techniques studied consistently detected small RNAs, but several miRNAs had considerably variable amounts. The relative relevance of minimizing the amount of total sequencing required, finding uncommon miRNAs, or absolute quantification should be considered while choosing the best methodology.

Many thanks dear Leonard Sebastian Fresenborg

Hey, just want to add to this even though this thread is pretty old. I thought the responses on here were thoughtful and super helpful so I'd like to chime in.

Nowadays, you can get most of your disposables Nuclease free because they gamma irradiate and pre-sterilize them.

you can buy water that is nuclease free.

as mentioned earlier, the older, sort of golden standard for water with RNA work was DEPC treated and that works fine for certain stains but for alot of downstream molecular biology applications are inhibited by depc.

I know people pulled off RNA research with less but if you can weasel in a budget for these, it might be a good move.

Also, I hear UV led lights are great for sterilizing pipettes, consumables, etc. we usually put our equipment under UV in a hood if we suspect strange results or contamination as a precaution. I'm a big fan of 10% bleach with di or milliq water to clean surfaces. I honestly avoid RNA zap and the like because it takes to look to dry and if I think my gloves are contaminated, I just change them for the sake of the experient.

just my 2 cents

RNA degrades if you look at it wrong.

Hello Antony, which protocols have you used? In all of them you get the same result shown in the photo?

Célia Bernard Because Aminoglycoside antibiotics promote misreading and inhibit the translocation of the tRNA-mRNA complex.

Thank you both, dear Paul Rutland and Keyla Santos Guedes de Sá

Thank you very much Laura Leighton an Deepdarshan Urs

Hello Ali Ghorbani

Have you referred to the protocol in the below attached paper?

If not, then you could try this protocol.

Best.

Thank you Dear Al-kuraishy

how about Sodium Acetate?

Serious question, have you tried calling their tech support? they are pretty helpful.

can I ask what your input is (how much tissue) and how much is your RNA yield? also, how fresh is your acetone?

Hi Roisin,

why not stick with Qiagen? To expensive?

Well, lysation may be the problem. Can you check the conditions (time, temperature, etc.) under the good old microscope to see if you need to change anything?

Best

Boris

Hi Marina,

Thanks for your reply. I think it's quite helpful.

Agniva Saha the problem was RNA integrity. I don't have my full protocol notes but briefly, cell lysis was performed in a 'gentle' buffer consisting of hypo-osmotic sucrose and 0.5% Triton X-100, agitated at a low temperature. I supplemented with superasin. Following lysis (which was incomplete after 30 minutes) the buffer was supplemented with 1.5% SDS and sodium chloride, Proteinase K was added, and Prot K digestion performed at room temperature for 45 minutes.

This whole protocol is a huge tradeoff between the needs of RNA (low temperature, rapid separation from other cellular components) and deproteination using Prot K (ideal reaction temperature 50-65C, and for deproteination of DNA after ChIP we do 4-hour reactions to get rid of all the protein.) The result was that I isolated less than 1/3 of the typical yield of low-integrity RNA (RIN ~5.5-6.5).

Compare this to phenol, which is such a strong protein and nucleic acid denaturant that it results in a) near complete lysis and b) near complete protection of RNA from endogenous RNases within seconds of efficient sample homogenisation. I don't really think there's an alternative that goes close.

I will say though, every method I mentioned above for avoiding phase separation results in no loss in RNA integrity if performed properly. I haven't done a head to head yield comparison, but my routine RNA extraction protocol involves Nucleozol supernatant mixed 1:1 with ethanol and run through a column, and the yields are better in my hands than alcohol precipitation of RNA, with better purity.

I find a lot of people don't realise quite how dilute cDNA can afford to be.

cDNA synthesis buffer itself is inhibitory to PCR (it contains DTT, for example), and you can also inhibit your reaction by saturating the system with too much target (or too much non-specific target).

In almost all cases, cDNA should be diluted prior to qPCR.

I typically dilute all my cDNA 1/20 (so for a 20ul prep I add 380ul water, giving me 400ul of final cDNA), and using 2ul of this per reaction is _plenty_: it allows me to reliably detect even low abundance genes.

For reference I use ~1600ng of RNA in a 20ul cDNA synthesis reaction, so 2ul of diluted stock is ~8ng of cDNA, assuming 1:1 conversion.

In your case it may be nothing to do with the lipids and everything to do with just...generally using too much cDNA.

When running dilution curves of cDNA historically, I expect the extremes at either end to be unreliable: with too much cDNA you have all the issues noted above, and with too little cDNA you have stochastic effects and thus highly variable data. This is FINE. The whole point of a dilution series of cDNA is to establish the range over which your reaction is linear and trustworthy.

Find that range, use it, don't worry about the extremes.

Artem Gorev Thank you so much for this! I completely missed this notification that you responded to my question. I ended up stumbling across the exact same PDF so I'm glad to see someone thinks the same as me. Thank you so much again!

Was the gut that didn't get homogenised effectively protected from RNases? If the leftover tissue was: stored in a reagent like RNAlater the whole time, OR frozen the whole time and never thawed, then I think it would be fine to extract the RNA from it now and add that RNA to the existing RNA from the same sample. But if the leftover tissue sat around in the collection tube at ambient temperatures for even a minute longer than the other samples - it's gone, let it go. Substantial differences in RNA quality between samples can be a major source of bias in RNAseq experiments, so deliberately adding RNA which is likely to be degraded is a bad idea. (If you are in doubt at all, do you have access to a BioAnalyzer or TapeStation or other way to check RNA integrity? You could extract the RNA from the leftover tissue, compare the RIN of the new extraction to the previous extraction, and mix them only if they are comparable.)

I don't think preparing a second library from the RNA made from the leftover tissue and then sequencing that would be worth the effort and expense. I don't think it would be possible to neatly integrate this data into your experiment.

Whatever you end up doing, this sample will be different in some way to the other samples in your experiment. So you should run an outlier analysis at the bioinformatic level, and seriously consider excluding this sample entirely if it looks different from the other samples. Building in some extra n into RNA-seq experiments to account for errors like this (they happen to us all!) is a very good idea when feasible.

if you have a budget go for small RNA sequencing and Transcriptome analysis.

follow the link file:///C:/Users/MLTFAC~1.NWI/AppData/Local Temp/QMF27.0254.M6955%20v3.1.pdf

Excessive homogenization or heat can be an issue. Check your storage of sample. You can use TE buffer and Store at -80. Samples stored more than a year under inappropriate conditions may lead to degradation. RNAse inhibitors can be used while extracting

I have used RNALater and the RLT+ buffer (part of the Qiagen RNeasy kit) to disperse and store cells in -80 for up to 6months (just haven't gone beyond 6months but don't see why it shouldn't work), you can place about 8million cells in 1ml of either and then pulse vortex for 30 seconds (no pulse vortexing does not degrade RNA, we have actually looked at this using Invitrogen's qubit IQ4 and the Agilent bioanalyzer 2100 to compare both RIN scores for RNA quality, in fact, vortexing leads to higher total RNA yield). Others here are correct in suggesting the RNeasy+. I also use Qiagen's RNeasy+ kits with the gDNA eliminator columns. Removal of DNA is a part of the kit so you don't need to add DNAse to a tube containing RNA or add that inactivating slurry later.

In that case Jessica Little the handbook on that page suggests 10exp8 cells should produce between 25 and 70ug RNA depending on the growth medium used so it looks like you can count cells,spin them down,decant most of the supenatant and then make up to a small convenient volume and add rnaprotect. You might contact the company making the rna for you and check with their tech support how and in what volume they want their samples sent. They will have a lot of expertise in converting various sample types and volumes to RNA

Dear Pia, thank you for sharing this interesting technical question with the RG community. Unfortunately I'm not a specialist in this field of research enough to give you a qualified answer because we are inorganic chemists. However, I know that the solutions to many technical problems can be found right here on RG. In this case, please have a look at the answers given to the following closely related question which has been asked earlier on RG:

(6 answers)

As you will see, other researchers already experienced the same problems. There is also the possibility to directly contact some of the RG members who provided answers to the 2017 question and ask them for some personal advice.

I hope this helps. Good luck with your work and best wishes, Frank edelmann

I am experiencing the same problem. I think it is not contamination but something with the oligo design.

Hi Annette, have you tried library preparation from heparin plasma? I am trying this now but unable to solve the problem.

Not an answer, but a suggestion:

How about rapid chilling of sample in liquid nitrogen or dry ice?

Think about it: plasma is usually at a near-constant 37 degrees. So whatever mRNA content it will contain will...probably be stable, at least in the short term, at anything at or below 37 degrees.

Thus: extract plasma as normal, using a room temp centrifuge, and then freeze the plasma. Freeze the plasma in modest aliquots (200ul is practical), so you don't need to keep freeze/thawing it. Store at -80.

Tris-buffer saturated phenol is quite different from Trizol. The essential component of Trizol is guanidinium thiocyanate, the very powerful сhaotropic agent, which dissociates nucleoproteins. If you add to the saturated phenol 1% of SDS, you receive the gold standard mixture for the RNA isolation.

Good luck!

Hi,

As I found out your Ct value is high, right?

There are many possibilities for the delay in Ct, the following solutions can be helpful:

1. Suitable concentration is 100 ng cDNA per 20 µL reaction. So, have you checked the quality and integrity of your extracted RNA using NanoDrops and agarose gel electrophoresis? Because you need to know your RNA concentration so that you can estimate your cDNA concentration in the next step.

Note: If the cDNA concentration is high, the concentration of inhibitors also increases and can result in a delay in Ct.

2. Suitable concentration of primers is 0.2 µM

If you tried these two and did not get the result, I suggest you change your materials. For example: using new master mix, sample re-extraction and ethanol percipitation, using RNA extraction kit and cDNA synthesis kit from other companies, using new master mix (from another company), etc

Most of the time when that happened to us. It was the reagent we used for blanking. What did you blank with? Guanidine will result in a massive increase A230, not reductions. So unless you blank it with something that has a trace amount of guanidine (or any other chemical that could interfere there) then measure your pure sample, you will have negative 230.

Mariana Batista Santos could you possibly send me your centrifuge speed and temperatures for each of the steps (your exact protocol)? I am using the same kit, and I am not sure on which centrifuge speed to start with, and I am short on time.

Hi,

I recommend using NanoDrops and agarose gel electrophoresis to check the quality and integrity of your extracted RNA sample, so you can use it if it has good quality and integrity.

Hi Dr David,

Thank you so much for your reply. Really appreciate your effort. I will read up and try out your suggestion! Thank you so much!

Missing Ct value can only be handled with a fresh PCR experiment. Many researchers use previous Ct values of housekeeping genes from their other experiments, however this is a bad practice and one must discourage this.

Hello,

I hope this will works with you.

Thanks.

The main consideration when choosing a lysis buffer is whether the chosen antibody will recognize denatured samples. When this is not the case, it will be noted on the antibody datasheet, and buffers without detergent or with relatively mild non-ionic detergents (NP-40, Triton X-100) should be used.

Katie A Burnette

We were just concerned about 2 bands so we did not use the ladder. We tried a board of conditions such as gel concentrations of 1, 1.5, and 2 %- formaldehyde and non-formaldehyde gel- TAE and TBE buffer- 100, 120, and 170 v for electrophoresis

Hi Mr. Abdullah,

In such situation, plasma collection and storage at sub zero temperature is the only option you have. You were already doing good.

Best wishes

Dear Piergiorgio,

thank you for sharing this very interesting technical question with the RG community. We work in the area of synthetic inorganic chemistry, so that I'm absolutely no expert in this area. However, I made the experience that it often pays off to directly search the "Publications" section of RG for potentially useful literature references. For example, the following detailed protocol is freely available as public full text on RG:

Good luck with your work and best wishes, Frank Edelmann

Qiagen has a RNA extraction kit specifically for tissue with a high lipid content (RNeasy Lipid Tissue Mini Kit) that might help maximize your RNA yield.

Sanaan Fathi thanks for your answer. We usually use Ethanol and Isopropanol in the RNA extraction protocol. The first one washing the resulted pellet and removing extraction solution or other contamination in the final step and the second one for precipitation of RNA molecules.

I think there is no problem in this serum preservation technique, because MiRNAs were considerably more resistant and has high stability than RNA.

Is your agarose actually boiling after heating. If the gel mix is just very hot the bubbles may be air being degassed from the buffer. Actually boiling the gel will degas the mixture and will minimise bubbling

Hi Keerthika,

How is your sample taken/stored? How did you homogenize your sample? Did you use trizol and chlorofrom? This is will help with removing the aqueous phase.

For future experiments- make sure your are cautious while removing the aqueous phase, using a 100ul pipette will help a lot.

Additional always use clean RNAse and DNAse free tubes for each step to avoid contamination. Use RNAzap if available to you between steps to clean your work bench and your hands frequently including pipettes (use barrier pipette tips if possible). Maintain temperatures as suggested by the kit/manufacturer throughout your experiment. You can also elude your sample in a smaller volume (try heating your water to 95c for elution).

YOu need to follow the manual protocol up to the chloroform step (DNA in aquous phase). then collect the aqueous phase and charge this on the kit column. this way you do not need to your the shredder column from the kit.

Omkar Anaspure Hi! Since you are using traditional method like Trizol, it is usual for concentration be high as 900 to even 2000 ng/μL. This high concentration is mainly due to presence of salts, carbohydrates and mainly proteins and peptides. That is the reason commercial kits obtain low but average concentration, since they purify all the unnecessary compounds (50 - 200 ng/ng/μL).

If you want to improve your sample by eliminating the impurities, I suggest you to use the commercially available spin column extraction method (silica based usually) so that your purification can be quick and very effective! The purification of your RNA will also increase your A 260/230 ratio (Keep it preferrably 1.9 to 2.2).

I can suggest you 3 methods for your further studies:

Hope you have your answer!

Nucleated cells. Mature mammalian RBC are not nucleated.

Dear Wei Li,

In addition to above suggestion:

1. Please use a density gradient separation medium with low endotoxin levels (such as Ficoll-Paque PLUS; density 1.077 g/mL) to separate PBMCs.

2. If feasible, use Qiagen's RNeasy Kit to isolate RNA from cells of your interest.

Hopefully, you will get the desired concentration of RNA with excellent 260/280 ratio.

Best wishes - Pradyumna

Now I use Polyacryl carrier (MRC) for total and small RNA precipitations, which works well even in recovering minute quantities. In the past, I used glycogen which worked great too.

The DNA contaminations can be easily removed by DNAse treatment.

Dear Johannes,

You are going to use the general protocol to isolate RNA. RNA isolation

Starting material: 1* 10^6 📷

Method:

A. miRNeasy mini kit (Qiagen), follow the manufacturer’s instructions.

B. General phenol-chloroform isolation method

1- put your samples in safe lock tubes (larvae sample)

2- Aspirate media and add appropriate amount of QIAzol to each well of cells , then collect your simples in safe lock tupes, and go to step 4.

3- Shake the tubes in the Bullet Blender speed 8 for 3 min

4- leave your samples 3 min on room temperature

5- Add 100 ul chloroform to the tubes and centrifuge them 15min- at 4 degree and 10,8 rcf

6- Remove the aqueous phase to new, fresh 1,5 tube

7- Add 100 ul 2- propanol and shake 15 sec (you will see a cloud like structure when RNA is present). To increase yield, samples can be keep at -20 for a couple of hours or overnight. This step is specially important for zebrafish larvae samples.

8- Centrifuge for 5 min at 4 c degree at maximum speed for pellet formation

9- Discard the solution and wash the pellet with 500ul- 750 ul Ethanol 70%

10-Centrifuge for 10 min at 4ᵒc at maximum speed. Then, a clear white pellet is visible

11-Remove the Ethanol till a small fraction is still covered in Ethanol. Then, Place the tube vertically

12- Let the remaining Ethanol airdry till the pellet no longer white but translucent

13- Add 25 uL RNA free water to the tubes and mix it thoroughly using the pipet.

Note: DNA removal is necessary for certain RNA applications that are sensitive to very small amounts of DNA (e.g., TaqMan RT-PCR analysis with a low-abundant target). Please, Follow the instruction in Appendix E: DNase Digestion of RNA before RNA Cleanup in RNeasy MINi kit.

14- Store your sample at -80ᵒc

Also check https://aip.scitation.org/doi/10.1063/1.5131795