RNA Extraction - Science method

Most qiagen RNA isolation kits include options for on-column DNAse treatment: it's not a huge effort (add DNAse to column, wash, continue with protocol) so that's an option if you're desperate.

Alternatively, why not simply determine whether you need to bother?

Most mtDNA encoded genes are expressed at super-high abundance, because they're fairly critical to cell function, so even in the presence of contaminating mtDNA, the cDNA signal should still represent the overwhelming majority.

Hence: prepare some "no reverse transcriptase" control samples: take RNA, prepare cDNA as usual but just...don't include reverse transcriptase.

Now run your qPCR with these and measure the difference between Cqs for "samples with RT, and same samples without".

If it's more than ~6 cycles, then honestly: ignore the fact you have some mtDNA contamination. 6 cycles is a ~64-fold difference, and thus mtDNA contributes only ~1.5% of your measured signal at best.

Since qPCR really isn't useful for measuring changes over such small scales, a random extra variable of +/- 1.5% is unlikely to be of consequence.

The kit was pretty user friendly but it didn't work well for my particular samples (leaves with high phenolic content).

NEB will often send a small sample-sized kit of many of their products. You could reach out to their customer service as well and ask if anyone has used it for your organism.

Dear Meng-Zhu Xie

The cDNA that you synthesized is for gene expression studies, so in such case, you should run comparative Ct protocol in RT-PCR for which you'll require house keeping genes as endogenous control. The Ct value of the endogenous control will be used to normalised your targetted gene, so no prior concentration of your cDNA is required.

Now if you want to analyze the yeild of your extracted RNA, you can run absolute quantification protocol using any known standards (now a days many standards are commercially available in some kits, like viral detection kit or NGS library quantification kits).

I did it for zebrafish larvae. The larvae were anesthetized, washed, and put in an Eppendorf tube with glass beads. The total RNA was isolated with a TRIzol reagent. The larvae were homogenized in the presence of TRIzol and glass beads, the chloroform was added and incubated for 3 min. After incubation, the mixture was centrifuged at 12,000 g for 15 min at 4’C. Three layered solutions formed, the upper clear portion containing RNA was pipetted to a new Eppendorf tube and ice-cold isopropanol was added and the tube was inverted several times to disperse any visible precipitate.

Daniel Martín Akshay Kumar thank you so much

Para mejorar la concentración de ARN en la extracción a partir de muestras de sangre en EDTA para metatranscriptómica, considera los siguientes pasos utilizando los reactivos de Qiagen mencionados: 1. **Lisis Celular Eficiente**: Asegúrate de que las células estén completamente lisadas. Puedes utilizar un homogeneizador o un kit de lisis específico para sangre si es necesario. 2. **Eliminación de Hemoglobina**: La hemoglobina puede interferir con la extracción de ARN. Utiliza un paso de eliminación de hemoglobina si es posible, como el que se incluye en algunos kits de Qiagen. 3. **Uso de Columnas de Captura**: Sigue las instrucciones del RNeasy Mini Kit para la captura de ARN. Asegúrate de lavar las columnas adecuadamente para eliminar impurezas. 4. **Elución Eficiente**: Utiliza un tampón de elución caliente (50-60°C) para mejorar la recuperación de ARN. Puedes aumentar la concentración de ARN eluyendo con volúmenes más pequeños (por ejemplo, 30-50 μL) y realizar múltiples eluciones. 5. **Control de Calidad**: Verifica la calidad y cantidad de ARN utilizando un espectrómetro de masas o un bioanalizador. Esto te permitirá ajustar los pasos de extracción si es necesario. 6. **Almacenamiento Adecuado**: Almacena el ARN a -80°C para evitar la degradación. 7. **Optimización de Volúmenes**: Ajusta los volúmenes de reactivos según las recomendaciones del fabricante para muestras con alto contenido de hemoglobina o células. 8. **Control de Contaminación por RNasas**: Asegúrate de que todos los reactivos y superficies estén libres de RNasas. 9. **Normalización de Muestras**: Si estás comparando muestras, asegúrate de normalizar las cantidades de ARN para análisis downstream. 10. **Repetibilidad**: Realiza extracciones en replicados para asegurar la consistencia de los resultados. Siguiendo estos pasos y optimizando cada etapa del proceso, deberías poder mejorar la concentración y calidad del ARN extraído para su análisis por metatranscriptómica.

Hi …

Here are some considerations:

Potential Options

_Contact Qiagen:The best approach would be to contact Qiagen directly. They can provide specific guidance on whether any modifications can be made or if there are any recommended workflows for your device.

_Modification: If you're technically inclined, you might explore whether you can modify the reagent tray or worktable to fit standard trays. However, this could void warranties or cause operational issues.

_Alternative Solutions: If conversion is not feasible, consider using the GeneRead QIACube for its intended purpose or investing in a standard QIACube designed specifically for nucleic acid isolation.

Dear Hamed Moslehi

Check the Human Protein Atlas for expression in tissues and cell lines.

You will see that highest expression level is in placenta and lung. I have no idea if you have access to these tissues.

The Human protein atlas also gives information about expression level in cell lines. Maybe, you have access to leukemia cell lines such as K-562. Lower expresison level is in SH-SY5Y, but this is a relatively common neuroblastoma cell line.

If you don't have access to these tissues or cell lines, you can get the sequence by gene synthesis. The region you are interested in is only approx. 200 aa, a gene synthesis for 600 bp is relatively cheap (should be less than 100 $).

Good luck,

Sebastian

Hi Manuela

expression of stress genes can be seen by this way, but one hour at room temperature even in the dark will conduct to most degradation of DNA and RNA. in addition to what you decided to do, I would add some storage in dry ice in an icebox for the travel.

best regards

fred

Bit late to respond here, but...are you just adding chloroform directly to the culture medium? That seems like a slightly odd thing to do.

As noted, chloroform is heavier than water (which culture media mostly is), and also does not mix with water, so...sinking to the bottom, leaving the media above, largely unaffected, is the expected outcome.

I would suggest you use a phenol/guanidium-based approach, like trizol: here the phenol WILL mix with the aqueous culture medium, and this plus the guanidium will lyse/denature/unfold anything unlucky enough to not be RNA. Do this, with enough trizol to make it work (i.e. 1ml trizol to 200ul media, or similar such ratios), and THEN initiate phase separation via chloroform. Phenol preferentially mixes with chloroform (which doesn't mix with water) so the mix separates out into distinct phases, and all your protein and DNA ends up in the phenol/chloroform phase (or at the interphase), and your RNA is left in the aqueous phase at the top.

So yeah: do that. Use trizol, and use lots of it. Ideally use the trizol/qiazol optimized for liquid samples (they make that).

I’ve had similar issues recently isolating small quantities of RNA (ribosome protected fragments). I use 300 mM sodium acetate pH 5.2 and 5 mM magnesium chloride followed by ethanol precipitation (2.5 volumes) overnight at -20 C with addition of 1.5 uL GlycoBlue. Spin at 20,000 x g for 1 h at 4 C in Eppendorf low bind tubes. I see good pellets in some samples but not for others. I do invert tubes sufficiently after addition of ethanol. I wonder if vortexing and centrifuging again might help. In some samples in looks like several particles precipitated on the side of the tube and not the bottom. Perhaps such multi-localized pelleting is the issue. Otherwise my best guess is RNase contamination. Although I am very careful with using fresh gloves and RNase Away on pipettes and gloves.

Según tengo entendido se usa el gel con TAE DEPC.

Hi Arbesu, I am wondering such RNA concentration of 15-20 ng/uL is enough to do cDNA Reverse Transcription? And from your experience, what ratio of A280/260 were you getting from Nanodrop of the platelet RNA sample? I appreciate any replies from you!

The reason you re getting less yield with gauge needles might be due to incomplete/partial mechanical breakdown. I see you said that you used 8 (perhaps you meant 18?), perhaps the 8 gauge isn't breaking down enough of the tissue clumps so that the 21 and 25 gauge can finish off at a finer level. I would stress that the first gauge is crucial for the success of the finer needles. 21 and 25 are quiet tough, I know 21 gauge was giving me real difficulty/strain on my fingers.

Colleen M Ingram We determined that it was not DNA contamination! We found an article on honeybees that saw a similar pattern. We figured out that the heating step of my RNA extraction kit was the problem, which looks like what is discussed in this publication! Thank you so much for sharing, I will definitely give this a read -- wish I could send it back in time to me a few years ago, haha. :')

Yes you could combine the RNA from different columns together.

The Qiagen kit also says you should get 10-20ug of RNA from lungs, assuming 30mg. However, the binding capacity for the column is 100ug. So in theory, you could use 150-300mg tissue and still be under the max binding capacity.

Depending on how the samples were frozen years ago, flash freeze in liquid nitrogen then store at -80 vs just putting them in the -80 to freeze, could definitely contribute to degradation.

I use 1ul of glycogen to pellet my RNA.

Hi Francisco,

I am also curious if you succeed with RNA isolation. I am having the same problem; my pellet is very viscous, and I am looking for a solution right now.

Thanks,

Tetiana

Hello,

In the context of RNA extraction, both phenol:chloroform:isoamyl alcohol and plain chloroform have been traditionally used, each serving specific roles in the process of isolating high-quality RNA.

In summary, both phenol:chloroform:isoamyl alcohol and plain chloroform have roles in RNA extraction, with the choice and use depending on the specific requirements of the RNA extraction protocol and the nature of the sample. Proper handling and adherence to protocols are essential for obtaining high-quality RNA suitable for downstream applications.

Check out this protocol list; it might provide additional insights for resolving the issue.

The presence of genomic DNA in the sample can be considered as a contaminant because of that during running Real time PCR, it affects quantity of cDNA (which is expected to come from expressed genes/transcripts). Since some DNA probes can bind to any double stranded DNA molecules, the presence of genomic DNA can affect quantification of cDNA.

Hello Esraa Mohsen

You will have to precipitate out the DNA from the lower, organic phenol-chloroform phase which contains both DNA as well as protein. The DNA may be precipitated out by the addition of ethanol, and the proteins may remain in the phenol-ethanol supernatant.

The paper attached below has demonstrated that the proteins isolated from samples stored as phenol-ethanol supernatants for up to 3 years at −20°C did not suffer from significant degradation as assessed by Western blot analysis.

The proteins may be recovered from phenol-ethanol supernatant by either precipitation or by dialysis, although the dialyzed pellets are significantly easier to solubilize and thereby result in better recovery efficiency. A combination of 0.05% SDS and 4 M urea in Tris-HCl, pH 8.0, will allow for complete solubilization of dialyzed pellets without apparent decrease in the integrity of the isolated proteins.

You may want to refer to the below attached article for more information.

Best.

hello sir, I can do it. RNA concentration in nonadrop was be 2107 ng/ul

Thank you so much

Have you tried a handheld homogenizer? I recently started using the benchmark D1000 to increase my RNA yield and it worked well.

Yes You can. Even the kit says the same can be applied for fungal extraction

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

Dear Paweł Małecki

Formamide-based RNA extraction protocols can indeed be difficult to find in the literature, but there are some strategies that you can try to concentrate your RNA after extraction.

One possible approach is ethanol precipitation. After extracting the RNA with formamide, you can add an equal volume of cold 100% ethanol and mix well. Then, centrifuge the sample at a high speed (e.g. 12,000-16,000 x g) for 15-30 minutes at 4°C. Carefully remove the supernatant, which should contain the majority of the formamide and other contaminants, and wash the RNA pellet with 70% ethanol to remove any remaining salts or other contaminants. After air-drying or briefly centrifuging the pellet to remove residual ethanol, you can resuspend the RNA in a smaller volume of nuclease-free water or buffer.

Another possible approach is LiCl precipitation. After extracting the RNA with formamide, you can add 2.5 volumes of 7.5 M LiCl and incubate the sample on ice for 30-60 minutes. Then, centrifuge the sample at a high speed (e.g. 12,000-16,000 x g) for 15-30 minutes at 4°C. Carefully remove the supernatant, which should contain the majority of the formamide and other contaminants, and wash the RNA pellet with 70% ethanol to remove any remaining salts or other contaminants. After air-drying or briefly centrifuging the pellet to remove residual ethanol, you can resuspend the RNA in a smaller volume of nuclease-free water or buffer.

Finally, you can try vacuum concentration using a SpeedVac or similar apparatus. While the boiling point of formamide is indeed high, it may be possible to evaporate off at least some of the formamide using a gentle vacuum and mild heat. However, you would need to be careful not to overheat the sample, as this could lead to RNA degradation.

Ultimately, the best approach will depend on the specific properties of your RNA and the contaminants present in your sample. You may need to try several different methods to find a protocol that works well for your particular situation.

Best Wishes

Hi,

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

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.

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.