Application Note

The regulatory effects microRNA of are widespread and microRNAs have recently been implicated in various diseases of the blood, stem cells, brain, and other tissues.

MicroRNAs are expressed in the blood

A study involving sickle cell diseases revealed that there is significant microRNA expression in red blood cells^[1] and another showed that microRNAs are highly expressed in the ischemic brain and can be detected in blood samples^[2].

MicroRNA expression analysis of blood samples can further our understanding of diseases of the blood

Analysis of microRNA expression in samples extracted from peripheral blood has shed some light on the causes of systemic lupus erythematosus^[3] and various aberrantly expressed microRNAs were detected in granulocytes, mononuclear cells, platelets, and reticulocytes of patients with polycythemia vera^[4].  Altered microRNA expression has also been detected in granulocytes isolated from blood samples of patients with primary myelofibrosis^[5]

MicroRNA extraction protocol for blood samples

There is a need to extract microRNA from whole blood samples for expression analysis to continue the research effort toward better understanding of these and other diseases.

Two important considerations when isolating RNA from whole blood samples are:  first, the sample collection method as well as the stability of the RNA must be considered.  It is very important to limit the time from sample collection to microRNA analysis.  Next, as with all sample types, it is very important to use a total RNA preparation procedure that does not remove the low molecular weight RNA fractions.

Several published studies^[6,7] have demonstrated good expression results when the PAXgene™ system is used as a blood sample collection method.  Although these studies made use of the manufacturer’s subsequent recommended RNA extraction procedure (or slight modification thereof) our extensive in-house development work has resulted in a proprietary alternative extraction method which we have used repeatedly to generate robust and reliable expression results.

LC Sciences’ recommended procedure for collecting blood samples for microRNA analysis:

1. Use Tempus™ Blood RNA Tube. (Life Technologies)
2. Collect multiple tubes from each donor and make note of the order of collection.
3. Keep the samples cold and limit the time to analysis. (follow manufacturer’s recommendation for storage times/temps)
4. Use at least two tubes from each donor. (Do not use the first tube collected from a donor)

LC Sciences’ recommended procedure for extracting RNA from blood samples for microRNA analysis:

1. Use an RNA Purification Kit designed to extract from blood samples stored in Tempus™ Tubes- Norgen Biotek
2. For extraction of microRNA from plasma samples we recommend the Norgen Biotek kit.

QC Note – You should expect to see lower than normal UV ratios for blood/plasma samples. Please contact us if you are unsure.  See the ordering page for more specifics on QC of your total RNA sample.

References

1. Chen SY, Wang Y, Telen MJ, Chi JT. (2008) The genomic analysis of erythrocyte microRNA expression in sickle cell diseases. PLoS ONE 4;3(6):e2360. [article]
2. Jeyaseelan K, Lim KY, Armugam A. (2008) MicroRNA expression in the blood and brain of rats subjected to transient focal ischemia by middle cerebral artery occlusion. Stroke 39(3):959-66. [article]
3. Dai Y, Huang YS, Tang M, Lv TY, Hu CX, Tan YH, Xu ZM, Yin YB. (2007) Microarray analysis of microRNA expression in peripheral blood cells of systemic lupus erythematosus patients. Lupus 16(12):939-46. [article]
4. Bruchova H, Merkerova M, Prchal JT. (2008) Aberrant expression of microRNA in polycythemia vera. Haematologica Jul;93(7):1009-16. [article]
5. Guglielmelli P, Tozzi L, Pancrazzi A, Bogani C, Antonioli E, Ponziani V, Poli G, Zini R, Ferrari S, Manfredini R, Bosi A, Vannucchi AM; MPD Research Consortium. (2007) MicroRNA expression profile in granulocytes from primary myelofibrosis patients. Exp Hematol 35(11):1708-18. [article]
6. Kruhøffer M, Dyrskjøt L, Voss T, Lindberg RL, Wyrich R, Thykjaer T, Orntoft TF. (2007) Isolation of microarray-grade total RNA, microRNA, and DNA from a single PAXgene blood RNA tube. J Mol Diagn 9(4):452-8. [article]
7. Liu J, Walter E, Stenger D, Thach D. (2006) Effects of globin mRNA reduction methods on gene expression profiles from whole blood. J Mol Diagn 8(5):551-8. [article]