Ghent University Isolates RNA in Dried Blood Microsamples

May 2024 — In a collaboration with Trajan Scientific and Medical (Trajan), researchers in the OncoRNALab at Ghent University in Belgium, conducted an RNA-based pilot study using blood microsamples to assess the performance of a new RNA-stabilizing agent.

The researchers used Neoteryx^® Mitra^® microsampling devices based on VAMS^® technology donated by Trajan to collect the blood microsamples.

These remote devices enable virtually anyone to easily collect blood samples from a quick finger-stick.

The samples can be mailed from remote locations to the lab via standard mail for analysis as dried specimens.

Prior to blood collection, OncoRNALab, Ghent University researcher Elena Ramos Varas, a PhD fellow working under Prof. Jo Vandesompele and Prof. Pieter Mesdagh, pre-treated the absorptive VAMS tips in each Mitra device with GenTegraRNA-NEO (GTRNEO-32).

GenTegraRNA-NEO is an Active Chemical Protection™ (ACP) technology by GenTegra.

Pre-treating the devices with the GenTegraRNA-NEO agent is designed to keep RNA stable in the blood samples for up to seven days at ambient temperatures, without the need for cold shipping or storage. 

The researchers assessed the efficiency of the GenTegraRNA-NEO (GTRNEO-32) for preserving RNA in whole blood microsamples over a period of 7-14 days. A Promega Maxwell RSC simplyRNA Kit was used in conjunction with the pre-treated blood microsampling devices to isolate RNA according to methods described in a Promega application note. 

RNA Pilot Study Design & Methods

To assess the performance of the RNA stabilizer, blood microsamples were collected from the same healthy volunteer using a 30 µL VAMS tip in a Mitra device.

• The microsamples were collected at three different time points before RNA isolation at 1, 7, and 14 days
  
  
• For each time point, 2 VAMS tips had been pre-treated with the stabilizing agent 24 hours in advance
  
  
• Additionally, at each time point, 3 microsamples with no RNA stabilizer were included as a control
  
  
• RNA concentration was measured using the NanoDrop (Thermo Scientific)
  
  
• RNA integrity was assessed using the FragmentAnalyzer
  
  
• RT-qPCR was conducted for blood abundant transcripts
  
  
• RNA-specific assays were used to evaluate RNA quality

RNA Pilot Study Results

• The group observed RNA degradation seven days after sample collection when GTRNEO-32 was not used to pre-treat the sampling devices
  
  
• Pre-treatment of the VAMS^® tips with GTRNEO-32 prevented RNA degradation
  
  
• With the pre-treated devices, the group could still isolate high-quality RNA 14 days after sample collection 

When using GTRNEO-32, RQN values remain stable 7 and 14 days after blood collection. (Image Credit: OncoRNALab)

RNA quantification by RT-qPCR

• RNA eluates were reverse transcribed with iScript Advanced cDNA Synthesis Kit for RT-qPCR (Bio-Rad, #1725037)
  
  
• qPCR was performed for genes that are highly expressed in blood (ACTB, HBA2, and HBB), using an assay that is RNA specific (TMED2, does not detect the DNA 
  counterpart)
  
  
• For all genes, Cq values were lower in the GTRNEO-32 treated microsamples than in the non-treated ones. This indicates that, with GTRNEO-32, higher target RNA levels are quantified.
  
  
• Some variability was observed across time points. There seems to be a tendency towards higher Cq values in samples stored for 7 days after blood collection than those only stored one day after microsampling.
  
  
• Surprisingly, microsamples stored for 14 days returned Cq values equal or lower than those stored for 7 days.

The OncoRNALab team hopes to publish their results and raise funding to launch a larger follow-up study using Mitra devices treated with GenTegraRNA-NEO to analyze RNA in up to 10,000 blood samples.

For more information about the work of the research team in the OncoRNALab at Ghent University, please visit their laboratory web page or list of publications.

Learn more about Mitra devices used with GenTegraRNA-NEO here.

Image Credits: OncoRNALab, Ghent University, Trajan