260/280 ratios are an unreliable and insensitive measure to assess purity (see also MIQE guidelines, Bustin et al., 2009). We also standardize our input based on similar volumes of serum for extraction and similar volumes of RNA in the RT reaction. By using the spike-in controls that Qiagen provides, you can assess extraction efficiency (C. elegans miR spike) and RT efficiency pointing at inhibition of the sample (miRTC spike). We accept variable levels of extraction, but no variance on RT efficiency. Differences in miR content are canceled out by the normalization process. We use the modified global mean normalization method (see attached paper; more papers on http://www.biogazelle.com/knowledge-center/publications). Biogazelle's qbase+ software can do this (and other) type(s) of normalization (http://www.biogazelle.com/qbase/key-features#normalization-strategies).

Article miRNA Expression Profiling: From Reference Genes to Global M...

Dear Sheyla,

You may try a different method for your measurements. A similar problem we also had in our lab, especially with frozen tissue samples with miRNA fraction. We tested the measured concentrations on the gel and we could only verify them when we measured with Ribogreen. Spectrophotometer results were varying and unstable, and depending on the Degradation rate they were misleading (although it works quite nice for total RNA). They also get iffy when the actual concentration is low (e.g. 3 sequential measurements can be: 15 ng - 35 ng -9 ng). In short, it might be the method that you use for measurement rather than your actual Situation raising the problem.

Spectrometric methods rather measure nucleotides than intact RNAs. When you work with miRNA fraction it is low concentration-small size, therefore the effect of Degradation products get higher. I cannot say much about your purity problem, but I guess it would also get better with Ribogreen (this was at least what we experienced).

Off note spectrophotometry worked very nicely for miRNA from cell lines.

Second, you might check other cDNA Synthesis kits with which you can convert from as less as 1 picogram of RNA (RevertAid from Thermo Fisher (previously Fermentas)), but of course this will increase you PCR cycle number for detection.

Good luck

Hi,

I observe often low 260/280 ratio when the concentration of the RNA solution is low. The Qubit/Ribogreen method is much more sensitive than the OD mesurement.

I am actually fighting with variable 260/230 ratio (The ratio is often bad when the concentration is low) of samples that I use in RT-qPCR. However, I don't have the impression that these variable ratio influence the quantification of my reference genes... Thus at your place I would try to work with these miRNA. If you really want to improve the ratio, make a phenol extraction, but this will worsen your 260/230 unless you work very clean and make sufficient chlorofom extraction, but in this case you'll probably lose to much material... Hope it helps!

Hi,

I obtained best results for extraction of total RNA with hot acid phenol. You can, like people said above, make another extraction with acid phenol and chloroform, and to finish, make an ethanol precipitation, just to concentrate your sample. If you want just make qPCR, you don't need absolutely a good ratio 260/280 or 260/230. good luck

Sheyla,

The comments above are all good. When we have trouble with RNA yield in our lab, I pool multiple samples together into one to increase the concentration in a standardized volume. It may be worth taking your final extract and doing one more acid phenol extraction to clean it up a bit. Precipitate the RNA from the final eluant volume using cold Na Acetate and cold ethanol (let sit in this solution for >5h prior to centrifugation to increase yields). Be careful about washing any precipitated RNA pellets with EtOH, as this will cause loss of yield. Best regards.

We use the miRneasy kit in our lab. For the majority of samples, quality and quantity were excellent. The step where you transfer the upper aqueous phase to the new tube, you must pay close attention not to contaminate your sample with the lower phase that contains Qiazol, DNA and other laysed components. I noticed that this stage is important and tricky, because you want to extract all of the miRNA found in the aqueous phase and once the tip touches the bottom layer, your sample is contaminated.

Jehan, which equipment did you use to measure the quality and quantity of your samples?

We use Nanodrop spectrophotometer to measure the quality and the quantity of RNA for our samples before proceeding with PCR

Serum contains low amount of RNA depending on the disease you are investigating. In cancer for example serum also contains RNases with variable amounts depending on the tumor stage and grade. Thus you have to obtain fresh samples and process them immediately. You have also to maintain RNase free environment for your work as possible as you deal with fragmented RNA liable to further degradation. You can quantify RNA using nanodrop instead of spec. and you can use larger volumes for extractions with smaller volumes of elution to increase the yield.

I would suggest that you read this journal article. It will give you some real food for thought about choosing an extraction method for your situation..Importance of RNA isolation methods for analysis of exosomal RNA: Evaluation of

different methods. Eldh et al. / Molecular Immunology 50 (2012) 278–286

260/280 ratios are an unreliable and insensitive measure to assess purity (see also MIQE guidelines, Bustin et al., 2009). We also standardize our input based on similar volumes of serum for extraction and similar volumes of RNA in the RT reaction. By using the spike-in controls that Qiagen provides, you can assess extraction efficiency (C. elegans miR spike) and RT efficiency pointing at inhibition of the sample (miRTC spike). We accept variable levels of extraction, but no variance on RT efficiency. Differences in miR content are canceled out by the normalization process. We use the modified global mean normalization method (see attached paper; more papers on http://www.biogazelle.com/knowledge-center/publications). Biogazelle's qbase+ software can do this (and other) type(s) of normalization (http://www.biogazelle.com/qbase/key-features#normalization-strategies).

Article miRNA Expression Profiling: From Reference Genes to Global M...

There's not much to add after Jo Vandesompele has weighed in, other than to emphasize the reliability of his recommendations! I would mention that confidence in even the spec concentration readouts would be low at the concentrations you mentioned. The goal of the study (biomarker discovery or otherwise) does not change the approach: use equal volume input with proper process controls and a global normalization method, as Jo mentioned.

i have same problem too

for same sample with different modification of trizol procedure .

i see these results : 

od : 1.302- 1.289- 1.339 - 1.24

concentration : 0.102 - 0.78- 0.099 - 0.178 micro-gram /ml

to day i run gel for last results but i didnt find any bond . is it normal???