Transcriptomics and RT-PCR relies upon the isolation of RNA, however RNA is relatively unstable compared to DNA due to differences in the chemical structure and the prevalence of RNases that degrade RNA. Labware can be purchased that is guaranteed RNase free, and specialized workflows employed to maximize RNA recovery from samples such as whole blood.
Such labware includes PAXgene Blood RNA Tubes by PreAnalytiX, and Thermo Fisher Applied Biosystems™ Tempus™ Blood RNA Tubes. RNA isolation kits include PaxGene® Blood miRNA Kit, MagMax for Stabilized Blood Tubes RNA Isolation Kit and Tempus™ Spin RNA Isolation Kit.
A recent study conducted by Jose Antonio Carrillo-Ávila of the Andalusian Public Health System Biobank, Granada, Spain, published in the journal BMC Research Notes, investigated RNA quality purified from 300 blood samples from 25 donors processed by two technicians using three methodologies using the Tempus and PaxGene tubes.
The best yields with a median of approximately 15µg were obtained using Tempus® tubes and the Tempus™ Spin RNA Isolation Kit. This method also had the joint-best A260/A280 ratio (least protein contamination) and the best A260/A230 (least organic contaminants).
RNA integrity (RIN) can be calculated using Agilent’s statistical model of RIN that takes account of the ratio between the 18S and 28S rRNA peaks in a standardized manner and assigns a value between 1-10, worst to best RNA integrity. Tempus® tubes and the Tempus™ Spin RNA Isolation Kit also produced the joint-best RIN.
Overall Tempus® tubes and the Tempus™ Spin RNA Isolation Kit results were the most reproducible. The PaxGene® Blood miRNA Kit notably produced the best results when specifically analyzing miRNA.
“To summarize, superior results were obtained with [PaxGene® tubes and the PaxGene® Blood miRNA Kit] and [Tempus® tubes and the Tempus™ Spin RNA Isolation Kit]. For Biobank large-cohort and epidemiological studies, [Tempus® tubes and the Tempus™ Spin RNA Isolation Kit] is the most appropriate methodology. However, the reduced hands-on time that is needed for [PaxGene® tubes and the PaxGene® Blood miRNA Kit] must also be considered,” concluded the authors.