Dear Prof. Jasmin Sutkovic,

It is not recommended to extract RNA from frozen blood samples. If the blood samples have been frozen, the thawing process is going to cause more cell damage. Cells will lyse from the shearing forces of ice crystals and from the shock to the cell membrane, and some of the cells will be lost.

Nevertheless, below are few tips for improvement.

1. First and foremost, you should collect blood in specialized storage tubes such as PAXgene, Tempus or RNA shield blood RNA tubes. These tubes contain reagents to ensure that intracellular RNA is stabilized effectively.

Blood plasma is high in RNase activity. RNase rapidly degrade RNA if whole blood samples are stored or processed incorrectly. This can change the RNA profile of whole blood samples within mins. Therefore, it is critical to immediately inhibit RNase activity upon collection of whole blood to ensure high quality RNA which is critical for downstream processing.

2. Quickly thawing frozen whole blood samples on aluminum blocks at room temperature could minimize RNA degradation, and improve RNA yield and quality compared with thawing the samples in a 37°C water bath.

3. To significantly improve RNA quality (RIN), mix TRIzol at 10:1 ratio (TRIzol:blood) to frozen blood so that the blood is in contact with TRIzol reagent as it thaws. Once thawed and mixed with TRIzol, wait and allow it to come to room temperature.

Regards,

Malcolm Nobre

I would try to incorporate a phenol-chloroform extraction step to the TRIZOL protocol, it should be able to enhance RNA recovery. However, I'd advise against using frozen blood samples, since cell damage prior to recovery could yield harder extractions.

Hope you the best,

Nicolas Xu

Jasmin Šutković did you mean you tried TRIZOL extraction with the column based extraction?