Effects of Peripheral Blood Different Pretreatment Methods and Preservation Time on RNA Quality

Abstract

ObjectiveTo evaluate the effects of different pretreatment methods and preservation time on RNA quality of peripheral blood samples, and to optimize the preservation method of peripheral blood samples.MethodsEight pretreatment methods were used to preprocess the peripheral blood from 3 healthy unrelated individuals and the treated samples were stored at -80 ℃. Total RNA of samples was extracted using Quick-RNATM Miniprep Plus kit. DNA/RNA ShieldTM was added to peripheral blood and total RNA was extracted after preservation at -80 ℃ for 0, 5, 10, 15, 30 and 60 days, respectively. The concentration, purity and integrity of RNA were determined. Statistical analyses were performed by SPSS 22.0 software to compare the differences in RNA yield, purity and integrity among the eight pretreatment methods.ResultsIn terms of purity, leukocyte pretreated with RNAlaterTM and directly cryopreservation peripheral blood showed the worst purity. The other six methods showed better purity. In terms of yield, blood cells with DNA/RNA ShieldTM came out with the highest yield, followed by peripheral blood with DNA/RNA ShieldTM. In terms of integrity, peripheral blood preserved in PAXgene Blood RNA tube method had the best integrity. Except for peripheral blood pretreated with DNA/RNA ShieldTM and blood cells pretreated with DNA/RNA shieldTM, the other five methods had statistical differences when compared to the method by keeping peripheral blood in PAXgene Blood RNA tube. The purity of RNA stored at six-time gradients ranged from 1.815 to 1.952. With the increase of storage time, RNA yield decreased from 4.516 ng to 1.039 ng, and RNA integrity decreased from 8.533 to 7.150.ConclusionAccording to the results of total RNA’s yield, purity and integrity, peripheral blood pretreated with DNA/RNA ShieldTM was the best pretreatment method. After the pretreatment, samples can be preserved for up to 60 days in low temperature.

Keywords

• forensic genetics
• ribonucleic acid （rna）
• pretreatment
• storage time
• peripheral blood