Stability of blood samples for analysis of tumour cell transcripts
(English)Manuscript (preprint) (Other academic)
When mRNA expression from tumor cells is analyzed in blood, one of the greatest problems is to keep the samples stable during transfer to the laboratory. Improved systems for stabilization of transcripts therefore have to be developed.
Blood was collected in PAXgene™ Blood RNA Tubes and spiked with melanoma and neuroblastoma cells. The stability of tumor transcripts at room temperature was tested after 1, 2, 3, 5, 7, and 9 days. PAXgene tubes were also frozen at -20 °C and -70 °C for 14 days. Total RNA was extracted by PAXgene™ Blood RNA Kit and mRNA was extracted by the GenoPrep Direct mRNA Kit. Tyrosinase, MAGE, tyrosine hydroxylase, and GD2 synthase mRNA was quantified by real-time PCR. The stability of these transcripts was compared with the stability in ACD tubes when RNA extraction was performed with QIAamp RNA Blood Mini Kit.
The yields of transcripts from the PAXgene tubes using the PAXgene™ Blood RNA Kit varied from 37% to 49% of the yields from ACD tubes. When mRNA was extracted with the GenoPrep Direct mRNA Kit the yields increased to 67-121%. Stability tests showed significant decreases of all the transcripts in the PAXgene tubes after two days at room temperature. After seven days, 10% of the initial transcript concentrations remained, whereas 32% remained in the ACD tubes. However, in the frozen PAXgene tubes the transcript levels were unchanged for 14 days.
We conclude that the PAXgene tube is suitable when the sample is stored at -20 °C or -70 °C and mRNA is extracted using GenoPrep Direct mRNA Kit.
RNA stability, real-time PCR, blood sampling, preanalytics, minimal residual disease
Medical and Health Sciences
IdentifiersURN: urn:nbn:se:liu:diva-84993OAI: oai:DiVA.org:liu-84993DiVA: diva2:563477