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Cell Free RNA Stabilisation

Cell Free RNA Stabilisation

mRNAs and microRNAs (miRNAs) have been identified in exosomes (nano-sized vesicles that are released from many cell types into the extracellular space and widely distributed in various body fluids). Recently, the use of microRNAs (miRNAs), such as exosomal microRNA-21 (miR-21) and Let7a as potential biomarkers was widely reported. Reviews entitled 'Considering Exosomal miR-21 as a Biomarker for Cancer, and Exosomal MicroRNA: Trafficking, Sorting, and Function summarise these findings.

Cell free RNA (Exosomal microRNAs) present in TransFix stabilised blood is retained for up to 10 days after stabilisation. Cell free RNA was detected by reverse transcription and qPCR, from purified exosomes and is detailed in the study below.

Case Study: Analysis of Cell Free RNA (Exosomal microRNA) by qPCR

On day 0 a blood draw from donors was performed. Each donor provided 3 x 9 ml of blood for both the conventional EDTA and TransFix/EDTA Vacuum Blood Collection Tubes (9ml tubes) from one venipuncture. The tubes were centrifuged at 300 x g for 20 minutes at room temperature. The upper plasma layer was separated and divided into 1 ml aliquots and samples were stored at 4°C.

On days 2, 4, 7 and 10 one tube of plasma from each donor was taken for exosome isolation, (using ExoCap™ Streptavidin Kit, MBL International). The isolated exosome-bead complex was frozen and stored at -20°C. Once the processing was completed for all 4 time-points, the isolated exosomes were analyzed by qPCR:

Reverse Transcription Protocol

  1. Thaw reverse transcription kit components on ice.
  2. Dilute reverse transcription primer to a 5X working stock solution using 0.1X TE buffer.
  3. Prepare reverse transcription master mix.
Component Master Mix Volume per 15 µL Reaction
100 mM dNTPs (with dTTP) 0.15 µL
MultiScribe reverse transcriptase, 50 U/µL 1.00 µL
10X reverse transcription buffer 1.50 µL
RNase inhibitor, 20 U/µL 0.19 µL
Nuclease-free water 4.16 µL
  1. Mix gently. Centrifuge to bring solution to the bottom of the tube.
  2. Place the reverse transcription master mix on ice.
  3. Vortex the reverse transcription primer tube, then centrifuge briefly.
  4. Aliquot 7 µL of reverse transcription master mix to 0.2 mL tube or 96 well reaction plate.
  5. Add 3 µL of 5X RT primer
  6. Add 5 µL RNA sample.
  7. Seal the tube or reaction plate and invert to mix the solution. Centrifuge briefly to bring the solution to the bottom of the tube or well.
  8. Incubate the tube or plate on ice for 5 minutes or until ready to load the thermal cycler.
  9. Run reverse transcription reaction in the thermal cycler.
Step Time (minutes) Temperature (°C)
Hold 30 16
Hold 30 42
Hold 5 85
Hold 4
  1. If you do not immediately continue to perform the PCR amplification after the reverse transcription reaction, store the reverse transcription reaction at -15 to -25 °

qPCR Amplification Protocol

  1. Thaw all components on ice.
  2. Mix gently. Centrifuge to bring solution to the bottom of the tube.
  3. Prepare the qPCR reaction mix in a 1.5 mL tube.
Component Master Mix Volume per 20 µL Reaction
TaqMan small RNA assay (20X) 1.00 µL
Production from reverse transcription reaction 1.33 µL
TaqMan universal PCR master mix II (2X), no UNG 10.00 µL
Nuclease-free water 7.67 µL
  1. Cap the tube and invert several times to mix. Centrifuge the tube briefly.
  2. Transfer 20 µL of the complete qPCR reaction mix to a 96 well plate.
  3. Seal the plate with an appropriate cover.
  4. Centrifuge the plate briefly.
  5. Load the plate on the PCR machine.
  6. Run the qPCR amplification.
Step Time Temperature (°C)
Hold 2 minutes 50
Hold 10 minutes 95
40 Cycles 15 seconds 95
40 Cycles 1 minute 60

Let7a qPCR Ct, -20°C Freeze/Thaw

Let7a qPCR Ct, -20°C Freeze/Thaw

miR21 qPCR Ct, -20°C Freeze/Thaw

miR21 qPCR Ct, -20°C Freeze/Thaw

Figure 1

Reverse transcription and qPCR miR21 and Let 7 analysis of patient samples shows the RNA integrity for all time points tested. This demonstrates that exosomes purified from normal serum collected into TransFix containing tubes and stored for up to 10 days at 4C yield miR21 and Let7a RNA that is suitable for amplification by qPCR, and shows measurable baseline amounts.

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