We extracted RNA from blood cells to analyze gene expression. We now have an aliquot with 500ng of the extracted RNA to make the reverse transcription (RT). The problem is that this 500ng are in a great variety of volumes. Some RNAs have a very low concentration (7ng/uL), in a way that if we use it like this till the end, we'll not have enough sample to detect in the qPCR.
First, we were told to dry the RNA in a vacuum chamber. However, when we talked to the lab that owns this chamber, they told us that the sample would heat during the process and that we would loose some RNA.
I talked then to the people from the company that makes the RT kit (High Capacity - Applied Biosystems). They told us that it would be better to make the RT with different RNA quantities and then adjust the cDNA after (in the vacuum chamber), to make the qPCR from the same amount of sample. They guaranteed that the RT reaction was linear and would not work differently for different amounts of RNA.
Another idea that was given was to "re-extract" the RNA, meaning that we would submit it again to a column and elute it in a smaller volume of water. The problem there is that too few liquid may not elute all the RNA, besides the fact that the kits are expensive.
We are not sure how to proceed and each person we asked told us something different. We don't want to loose much sample but, more important, to alter the qPCR results.
I attached a file with some examples of RNA concentration we have, to give a better idea.
Any thoughts on how we should proceed?
Thanks.
You can indeed concentrate your RNA by putting it in a vacuum chamber and heat it to 45 degrees Celsius. You dry your RNA until a pellet remains, this you can dissolve in the correct amount of RNAse-free water. I do this often with samples that have a low RNA concentration and never lost RNA and never witnessed any degradation due to this process.
Another option would just be a simple precipitation with 2.5 volume 100% Ethanol and 1/10 volume Sodiumacetate (3M, pH5.2). With these low concentrations add a carrier like glycogen.
I wouldn't do a re-extraction on a column as you will loose quite some RNA.
From your extraction, we have a complex of ribosome, mRNA, viral RNA... then you need to point out which RNA you need to analyse. Making cDNA is not simple 100% efficacy, you may deal with 3rd-4th dimension construction of RNA.
if your target is qPCR, it is better to optimize with one-step RT-PCR, and making serial dilution of sample to get best result. And make sure develop IC with a house-keeping gene to monitor all procedure.
If you make a bank of cDNA, then try to make a large volume assay (50-100ul), with heating primer/sample mix 60-70oC. And also try a cocktail of several enzyme.
May Be i Didn't fully understand your question , but what i understood according to that i would recommend this:
make the final working volume of RNA constant say for example you get 10 ng/uL (sample 1) and in other sample you got 5ng/uL(sample 2) . you require final working volume say 10 ng in a reaction than add 1 uL of sample 1 in aparticular reaction and 2 uL of sample 2 in a particular reaction. in this way you add same concentration of RNA.
as you are saying that other RNA samples are too concentrated dilute them by adding your elution buffer or Nuclease free DEPC treated sample.
All the best
You can indeed concentrate your RNA by putting it in a vacuum chamber and heat it to 45 degrees Celsius. You dry your RNA until a pellet remains, this you can dissolve in the correct amount of RNAse-free water. I do this often with samples that have a low RNA concentration and never lost RNA and never witnessed any degradation due to this process.
Another option would just be a simple precipitation with 2.5 volume 100% Ethanol and 1/10 volume Sodiumacetate (3M, pH5.2). With these low concentrations add a carrier like glycogen.
I wouldn't do a re-extraction on a column as you will loose quite some RNA.
Thanks for the answers. I may not have been so clear in my question... The issue is that if I use the recommended 10uL of RNA in the RT reaction of the least concentrated sample, I would be putting only 70ng of RNA to the reaction. Additionally, I would have to add 2.8uL of this cDNA to have something around 10ng in my 10uL qPCR.
I could dilute all my RNAs to work in this low concentrations, but some people told me I should not do an RT reaction with less than 300ng of RNA. For this same RNA, with 7ng/uL, I would need 42uL in my RT, but the limit recommended is 10uL.
I want to know what is less "harmful" for my qPCR of some target mRNAs. Should I work with low concentrations and make more RT reactions? Should I concentrate my RNAs before the RT? Or should I do the RT with different amounts of RNA for the different samples and then concentrate my cDNA (assuming that the cDNA amount is proportional to the RNA amount used)?
Alex, I am glad to hear you didn't experience loss of RNA by concentrating it. I will consider this in my decision. :)
In my opinion the best approach would be to concentrate your RNA by Ethanol precipitation, this would also be a further cleaning step. You should add 10 ug of glycogen (glycogen can inhibit the RT, but in concentration of >2 ug/ul, while even using all your RNA in a 20 ul RT reaction you will be at 0.5 ug/ul, so no worry), add NaOAc as suggested (you can also use final 0.2 M for RNA, you will have less salt precipitating, which is better for the RT), mix very well and then add the 2.5 vol. of EtOH. Mix well again by inversion and place at -80 °C or in dry ice, until the solution freezes, this will maximize precipitation. Normally a 10-15 min spin is more than enough to pull down all your RNA. You should obtain a clearly visible pellet due to the glycogen, although sometimes the precipitate smears on the back of the tube, but it's fine, it's still precipitated. Once you dry it in the air, you can easily solubilize it in 10 ul of H2O or 10 mM Tris/HCl pH 7.4 and proceed with the RT.
Good luck!
If your sample is in water why not simply freeze dry it (lyophilize)? If you don't have access to this equipment, then I would suggest Ethanol/Ammonium Acetate precipitation (2.5 Vol Ethanol/2.5M Ammonium Acetate final conc). with 10ug Glycogen as a carrier
Leave at -70C for 60 min and then spin 30 min at 4C. Aspirate off the supernatant and wash the pellet with ice cold 70% ethanol to remove the Ammonium Acetate. Then resuspend in an appropriate quantity of sterile H2O (assume you recover ~70-80% of your initial input RNA). Ammonium ions are easier to remove than Sodium and are less inhibitory to the RT reaction
These are the following points I can think of which may be of your help.
1) Over the period I realized that RNA isolation from Trizol reagent is much more efficient then RNA isolation kits. This will give you RNA with high concentration.
2) The concentration which you mentioned is very low for lyophilisation or precipitation. You may again land up in loosing RNA.
Wishing you all the best.
If you do decide to concentrate via precipitation you might try linear polyacrylamide as a carrier. It has several advantages over, say, glycogen. Sigma sells it or you can make it yourself with little trouble.
Which ever method you use, I would suggest practicing the technique first to see which one gives you the highest yield. Can you extract more RNA from other cells for practice? My experience with all of these methods is that you will lose some RNA. It happens even just from defrosting.
I dont know which blood cells did you work with and first of all you should enumerate the tcollected blood cells with haemocytometer, if its cultured blood cells, you can pool and extract RNA. You can easily get enough RNA for c DNA synthesis.You can pool the blood samples of same type or for same experiment.
For concentrating the RNA, I may recomend you to do the follwing:
perceptate the RNA with equal volume of isopropanol , incubate on ice for 10 min,centrifuge for 10 min at 7500rpm
then wash with about 500 ul of 70%ethanol (diluted with DEPC treated H2O),centrifuge for 15min at 12000rpm.
Remve ethanol,leave the RNA pellete to dry on RT for 10 min. Then resuspend youe RNA in the apropriate volume of buffer or DEPC treated H2O.
This is the last steps of triazol extraction and it is very efficient method in RNA extraction .
I realize this is pretty late, but I needed to put in my two bits. Since you are using this sample for qPCR it is pretty important that you keep your samples as highly pure as possible and devoid of carryover contaminants like salts, ethanol and carriers, because the quality of your RNA affects the quality of your cDNA and the Quality of your PCR, and therefore your resultant answer by qPCR. I would therefor either follow the manufactures recommendations and make the cDNA from the same volume of RNA and then use a speed-vac to dry down the lower concentration samples to the same relavent concentration of starting RNA. This may require you to make several cDNA reactions for some of your lower samples so that they can be combined and dried down together. For instance if your top sample is 100ng/ul and the cDNA reaction final volume is 20ul, then for your 7ng/ul sample you would need to dry it down to 7/100 x 20 = 1.4ul, so you would likely want to use more than one reaction so that you have closer to 10ul. It is likely that you will need to dilute your cDNA prior to using it in your qPCR, so this will affect how much sample you will need to generate. If not this, and you want to concentrate your RNA first, then Thermo Scientific has a wonderful product...GeneJet RNA Clean-up and Concentration Micro Kit...We had to use this kit for a couple of our samples, and I was super impressed by the level of recovery we got... close to 95% of the sample that was applied. They were recently giving out free 6 prep samples of this kit (and may still be).
Good Luck
Ron
Refrigerated vacuum concentrator is best option for concentrate the RNA and DNA at lower temperature
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