Your RNA quantities are fine and, contrary to popular opinion, anything greater than 1ug in a cDNA synthesis reaction is really unnecessary (5ug is ridiculous!!). In fact cDNA made with 50-100ng RNA in total will yield accurate and precise data in QRT-PCR assays. On quality, RNA should always give a 260/280 ratio >2.0 and as such your samples could be slightly suboptimal. Ratios of

I suppose, you did a total RNA extraction? 200ng x 50 is 10 ug, which is OK. Normally, we use 1-2 ug of total RNA to make cDNA in a volume of 10-20 uL. Depending on the expression of your gene of interest, you either make a dilution or not from this material to do a PCR. Anyway, that should be more then enough. About this ration, in my opinion if it is close to 2, it should be OK. Anyway, when you make cDNA for your RT, the quality of it will be the same for both your experimental gene and your reference gene.

Good luck

For the RT reaction, I use 50 ng/uL of total RNA in a final volume of 20 uL. A good 260/280 ratio is between 1.9 and 2.1, so I think your samples are ok. Remember also that this ratio only gives you an indication of RNA purity, so you will have to check its integrity by other method (for example, by denaturing agarose gel electrophoresis).

As Alex stated, RNA concentrations and 260/280 ratio is fine (2.2 to 1.8 is ok).

Contamination issues could be simply seen by looking at the measurement curves in nanodrop. For a clean sample you should get a maximum at 260 and a minimum at 230. By looking at 260/230 ratio you can also get an idea about purity (bigger than 1.7 is good). For contaminants like dsDNA or ssDNA you can't say anything with spectrometers since both will give a peak at 260.

However, as my experience of RNA extraction running by RNAeasy KIT, the A260/280 is always between 2-2.1. The concentration was OK for 200 ng/ul, it's enough to proceed into cDNA synthesis. How about the RT-PCR result?

I don't think you will face any problem in cDNA synthesis with this much amount of RNA concentration i.e. 200 ng/ul . In my opinion this is perfectly ok. Sometime we did our work with 20 ng/ul concentration even and got perfect results. So as per concentration you need not to worry much. As per purity of your RNA yes this is on lower side as nearby 2 is always good... but I think still you can proceed with this as this is in acceptable range of 1.8 to 2.2 and also some of your samples are having a ratio of 1.9 and 1.89 , remaining one are 1.85, 1.84 etc . so I don't think you will face much problem in your experiments. Good luck

I can only second Manmeets post. 200ng/uL is an ok concentration. I often work with 20ng/uL and have no problems. But if your RNA concentration is too low also depends a bit on sample size and purification method and sample (cell or tissue type).

Thank you all for such nice suggestions and also thanks this platform that helps a lot for young researcher around the world irrespective of country and thus disseminating the knowledge to build a better world. @ Alex, its total RNA extraction. @Stefano, i followed the Trizol method. @ Manmeet and Stefano, what do you think about 260/230 ratio as 4 samples ratio is below 1.3.

one more point, usually how much time do you take for RNA isolation? Do you run RT-PCR in the same day?

time taken during RNA isolation is really a important issue, although it depends upon how many samples ur handling at a time. I preferably use Qiagen kit so usually for 5-6 samples I do in 20-25 min. Regarding RT-PCR I don't think its an issue once you are done with your cDNA, its all safe. Yes I prefer cDNA synthesis on the same day, although you can store your RNA also in -80. Once you are with your cDNA you are perfectly ok. I usually do RT-PCR on next day.

Hello there, you can use as low as 50 ng of RNA for RT-PCR. I guess 200 ng is too much. Hope this helps.

Regarding low 260/230 ratios. Vi had that problem with trizol extraction from liver samples, but not other tissues. We found that two 70% eth-OH washes solved this issue.

With low conc. RNA purified with RNeasy we also encounter low 260/230 ratios but have not really found an easy solution for this except being extra careful to reduce RLT buffer crystals as much as possible, but still ratios are often just 1.3-1.7. However, RT and qPCR does not seem to be affected, there is no correlation between CT and 260/230 ratio.

the concentration that you have will pose no problem for RT PCR nor will the ratios. In my expereience the ratios depend on the starting material for isolation, for something which might have a lot of pollysaccharide I have found teh ratio to be lower. I would start worrying if the 260/280 ratios were 1.5 or less

I have worked with much lower concentrations. It works in most of the cases. In special circumI tried the RNeasy mini, and TRIZOL method, and nowdays I use mRNA isolation with magnetic beads. In last case, even if the concentration is too low (0,05μg), after cDNA synthesis the qPCR data for many housekeeping genes are ok.

Nucleic acids and proteins have absorbance maxima at 260 and

280 nm, respectively. Historically, the ratio of absorbances at

these wavelengths has been used as a measure of purity in both

nucleic acid and protein extractions. A ratio of ~1.8 is

generally accepted as “pure” for DNA; a ratio of ~2.0 is

generally accepted as “pure” for RNA.

260/280 Ratios

Abnormal 260/280 ratios usually indicate that the sample is

either contaminated by protein or a reagent such as phenol or

that there was an issue with the measurement.

A low A260/A280 ratio may be caused by:

• Residual phenol or other reagent associated with the

extraction protocol

• A very low concentration( > 10 ng/ul).of nucleic acid

High 260/280 purity ratios are not indicative of an issue.

Although purity ratios and spectral profiles are important

indicators of sample quality, the best indicator of DNA or RNA

quality is functionality in the downstream application of

interest. If the purity ratio is significantly higher than

expected, it is best to review the spectral profile as a primary

means of troubleshooting.

It is important to note that there are occasions when the purity

ratios are within expected limits, yet there is a problem with

the sample.

Your RNA quantities are fine and, contrary to popular opinion, anything greater than 1ug in a cDNA synthesis reaction is really unnecessary (5ug is ridiculous!!). In fact cDNA made with 50-100ng RNA in total will yield accurate and precise data in QRT-PCR assays. On quality, RNA should always give a 260/280 ratio >2.0 and as such your samples could be slightly suboptimal. Ratios of

If you read the manual of some cDNA kits, they will tell you 0.1 ng to 10 ug will all be fine.

I'm aware of the limitations and advantages of using the Nanodrop or the Qubit to determine the concentration of isolated RNA/DNA for further Gene Expression Assays by qRT-PCR.  I also use the Bioanalyzer (Agilent) to confirm my isolated RNA's integrity.   So, can anyone tell me, without describing in detail, what each, the Nanodrop or Qubit can do, what machine should I use to determine the concentration of RNA that I can use to make cDNA for qRT-PCR?  Like me, there are many investigators finding discrepancies in the concentrations of RNA using these two machines for reasons well known to us, but again.  Which machine is better to use in this case? By the way I isolate RNA from mouse brain.

Thank you!

Thanks everyone. This thread answered all of my questions in mind. I also got a very low RNA yield and was wondering if it will be enough for cDNA synthesis. I used TapeStation to view the quality of my RNA samples. I'm gonna try nanoDrop to know the A260/280 and A/260/230 ratios. I did TrizolLS extraction of my exosome samples and then passed it through miRNEasy kit to isolate the RNA. For some odd reason I saw that my RNA samples contain some contamination. I'm not sure if it's DNA, protein or chloroform? I'm hoping to have a better idea after nanodrop.

My RNA was accidentally diluted to 10 ug/ul, is there any chance the cDNA synthesis and qPCR could work? 

The nanodrop values are not always trustful to understand integrity of RNA samples. 

If I were you, I also check the integrity of RNA samples on agarose gel. If you see two intact bands on gel, you can work for RT-PCR. Good luck! 

https://www.thermofisher.com/content/dam/LifeTech/global/life-sciences/DNARNAPurification/Images/1114/f00286.gif

 @anna how many ul RNA do you have?

I always try to use 1ug of RNA for cDNA prep before I run my qRT-PCR. At this time, I have less amount of RNA, and I will try lower amount of RNA such as 0.5ug or 0.25ug. According to cDNA protocol of Maxima Thermo Fisher it should work:)

Good luck for all....

Hi Mikael, have you done it? I will check that, thanks...

Hi respected researchers,

I am working on RNA but my 260/ 280 ratio comes very low ( eg. 1.25 to 1.45) can i proceed my sample to RT PCR with this low purity Ratio of RNA..?

Hi Muhammad,

As long as you are working with RNA, the 260/280 ratio should be close to 2 to proceed with RT PCR. However, I run samples in RT-PCR with 1.8 and it was still fine but I think your purity is quite low so I wouldn't proceed further.