in the elution step: close the tube, leave the sample at 70ºC, for 5 min, them centrifuge and repeat this step one more time. collect both eluted samples in the same tube.

Thank you, Fernando! As I understand, RNA samples are contaminated with residuals of RLT buffer, in particular with guanidine isothiocyanate. So could you please tell me how can heating remove the contamination? Heating inactivate guanidine isothiocyanate or make anything else?

you are right about guanidine. In any case your RNA quality is good (RIN 5-8) so I think you can try your downstream application to test if the guanidine is interfering, usually it does not unless you add a lot.

good luck!

the heating is to remove RNA from the silica in the last step. To remove the guanidine isothiocyanate. you must do another washing in the washing step.

Thanks, Alfonso! We tried to use these RNA samples in microarray expression analysis (Agilent) and found that labeling with Cy3 failed. In contrast, RNA (RIN 6.0, 260/280 - 2.0, 260/230 - 1.8) isolated from tumor bulk was perfectly labeled. So, I think that low 260/280 and 260/230 ratios of RNA samples from microdissected samples are the cause of failed labeling.

In other applications, for example in RT PCR, this RNA works good.

Thanks, Fernando! I will try to repeat washing, although it can reduce RNA yield.

Have you tried isopropanol precipitation followed by phenol:chloroform? if you have enough after doing this it will be very clean.

I am afraid that isopropanol precipitation can dramatically reduce my RNA yield. Although I can add NaAc and glycogen to get better precipitation. Thanks for the suggestion!

I tried to purify RNA samples both with additional washing by RPE buffer and with ethanol precipitation as recommended by Thermo Scientific (http://www.thermoscientificbio.com/uploadedFiles/Resources/r055-product-information.pdf). In all cases, 260/280 and 260/230 ratios almost did not differ from initial ones. So, I may conclude that low 260/280 and 260/230 ratios are typical for low concentration of RNA samples, as previously suggested in other questions of Researchgate.

Dear evgeny

I would like to share my experience with RNA isolation from pbmc cells of 2-3ml of venous blood sample. I had tried both trizol method and Qiagen rna easy mini kit. But I did not get good yield as well as expected 260/280 & 260/280 ratio. So I combined both the method.In the first step; I treated the cells with trizol chloroform. After centrifugation(12000gx15 min) carefully removed the upper clear supernatent and used in RNa easy mini kit. Now I getting good results.

All the very best.

Thank you! I will try this protocol and will write here about results.

I tried to purify RNA with trizol chloroform followed by RNeasy Micro Kit, and RNA samples also had low 260/280 & 260/280 ratios.