TITLE: RNA and gDNA Isolation from stabilized Whole Blood (Tempus Blood RNA Tube) using semi-automated Promega Chemistry
AUTHORS: Merz MP, Treue D, Hummel M, Stege A, ZeBanC - Zentrale Biobank Charité

[DESCRIPTION]
Here we describe a semi-automated way to isolate RNA and gDNA from (the same) TEMPUS Tube. This allows the user to expand the use of TEMPUS Tubes (designed for RNA stability only) as well as describing a high throughput, highly reprudicible, semi-automated way using liquid handlers in the isolation process.

[BEFORE_START]
Materials to Be Supplied by the User:
• vortex mixer
• 50ml tubes (sterile)
• centrifuge with swinging-bucket rotor
• RNase-free, sterile, aerosol-resistant pipette tips
• Maxwell‱ RSC simplyRNA Blood Kit (Cat.# AS1380)
• ReliaPrep‱ Large Volume HT gDNA Isolation System (Cat.#A2751)

[GUIDELINES]
The Central Biobank Charite (ZeBanC) works under the best practice laboratory guidelines and ISO:9001 quality standards.
This protocol uses readily available Kits (by PromegaTM) in a semi-automated fashion. While we believe that, a similar worflow can be adapted for different manufacturers liquid-handlers, this protocol in its published form has only been validated on machines and chemicals as described here.
In case of doubt, please feel free to consult attached manufacturers SOPs or reach out to us.

[STEPS]
SECTION: Blood draw, tube handling and storage
1. Draw up to 3 mL of blood directly into a Tempus™ Blood RNA Tube according to your laboratory's or hospital standard. See manufacurers SOP for more information.


 

 

SECTION: Blood draw, tube handling and storage
2. Immediately after filling the tube, shake the tube vigorously or vortex the contents for 10 sec to ensure that the stabilizing reagent is thoroughly mixed with the sample. 
 

SECTION: Blood draw, tube handling and storage
3. Filled Tempus tubes should be shipped at room temperature (18-25°C) within a maximum of 5 days. Please consult manufacturer instructions for details.


SECTION: Blood draw, tube handling and storage
4. Filled Tempus can be frozen and stored at -20°C to -80°C until nucleic acid isolation.
 

SECTION: RNA isolation from Tempus tubes
5. Solution Preparation
This can be done in advance. Handle in fume hood!


Prepare (1-Thioglycerol) Homogenisation buffer (we do it for a batch of 32 samples)

12 mL 240 µL 
Stable at 2-10°C for up to 30 days.


Prepare  Rehydrate DNase-I solution (28 samples/tube) according to the manual.
Dissolve lypholysed DNase-I in 275 µL and add 5 µL
Should be stored at -20°C until use. Avoid repeated thaw-freeze cycles.

SECTION: gDNA Isolation from supernatant
7. On a TECAN Evo Freedom Instrument, run the following protocol on 8–10 ml of the supernatants from step 6.5. This protocol is fully automated (steps 7.2-7.13) and normally does not require hands-on interventions.
Other automated systems (i.e. Hamilton Microlab STAR, PerkinElmer JANUS etc) should be able to run the same or a similar protocol.

 

 
 
  
 

 


SECTION: RNA isolation from Tempus tubes
6. Thaw (32) Tempus tubes at room temperature (20-22°C). This will take approximately 30-60min.

 

SECTION: RNA isolation from Tempus tubes
6.1. Label a clean 50 mL (Falcon) tube with the sample identification.
Pour the entire volume of a Tempus tube (approx. 9 mL) into a fresh 50 mL (Falcon) tube
SECTION: RNA isolation from Tempus tubes
6.2. Use 1X PBS (phosphate-buffered saline) to a total volume of 12 mL.

 

SECTION: RNA isolation from Tempus tubes
6.3. Vortex for 30-45 sec at highest power.
 Make surethat the tube is securely capped. Frothing of the sample after vortexing is normal.



SECTION: RNA isolation from Tempus tubes
6.4. Centrifuge samples 4000 x g, 60 min, 4 °C 

SECTION: RNA isolation from Tempus tubes
6.5. Carefully pour the supernatant from each tube into a new 50 mL tube (keep new tube for later gDNA isolation). Do so in one movement, in order to not dislodge the RNA pellet. 

 


Use a paper towel to take off drops from the rim of the RNA 50 mL tube. 
Starting the next step with a minimal amount of supernatant is essential, therefore try to drain as much buffer as possible without over-drying or disturbing the pellet.

 


 


SECTION: RNA isolation from Tempus tubes
6.6. Add 300 µL (at RT) to every tube and resuspend pellet by vortexing (15sec).

SECTION: RNA isolation from Tempus tubes
6.7. Add  200 µL and 25 µL  per sample, vortex very shortly (approx 1 sec) and incubate at RT for 10min.

SECTION: RNA isolation from Tempus tubes
6.8. Transfer complete lysate into the first well of the Maxwell Cartridge (Promega # AS138A). Load the rest of the cartridge according to the Maxwell‱ RSC simplyRNA Blood Kit (#AS1380) instructions.
Add 10 µL to well number 4 (shown in yellow).
Place a 0.5 ml tube (part of Promega Kit #AS1380) in the corresponding slot on the Maxwell® RSC tray (see figure, position 2). Label this tube according to your samples. Add 65 µL  into the 0,5 ml tube.

 

SECTION: gDNA Isolation from supernatant
7.1. Load the instrument with bulk reagents (see table). 

 
SECTION: gDNA Isolation from supernatant
7.2. In a first step the actual volume of the sample is measured in order to calculate the volumes of subsequent reagents.
 
SECTION: gDNA Isolation from supernatant
7.3. Proteinase K Working Solution (0.02volumes; 60-90µL, based on the starting sample volume) is added to each tube and shaken at 500rpm, 25°C for 1 min.
SECTION: gDNA Isolation from supernatant
7.5. 1volume (3-5mL) of Lysis Buffer is added to each sample.
SECTION: gDNA Isolation from supernatant
7.4. Alkaline Protease (0.125volumes; 375-600µL) is added to each sample and shaken at 500rpm, 25°C for 1 min.
SECTION: gDNA Isolation from supernatant
7.6. After Lysis Buffer is added, the samples are incubated at 65°C for 30 minutes while shaking at 500rpm, followed by 10 minutes of shaking at 500rpm at 25°C. 
SECTION: gDNA Isolation from supernatant
7.7. Next, 1.2volumes (3600 - 5600µL) of Binding Buffer is added to each sample and shaken at 500rpm, 25°C for 3 min.
SECTION: gDNA Isolation from supernatant
7.8. ReliaPrep™ Resin is thoroughly mixed and 0.1volumes (300-500µL) of resin is added to each sample. Binding of gDNA to the resin happens during incubation at 25°C for 20 minutes at 550rpm followed by magnetic capture for 20 minutes to collect the resin.
SECTION: gDNA Isolation from supernatant
7.9. Waste from the lysis and binding is removed (by vacuum pump), then 5-7ml of Wash Buffer is added to each tube. The magnet then releases the resin into the Wash Buffer.
SECTION: gDNA Isolation from supernatant
7.10. Samples are shaken at 500rpm, 25°C, for 2 minutes. Afterwards they are tip-mixed to thoroughly disperse the resin. Following this, the instrument mixes by shaking at 500rpm for 2 more minutes. 
SECTION: gDNA Isolation from supernatant
7.11. Waste from the first wash is removed from each tube, and 4-9ml of Wash Buffer is added (based on the original sample volume) while shaking at 500rpm for 4 more minutes.
SECTION: gDNA Isolation from supernatant
7.12. Next, the resin is mangnetically captured for 3 minutes, the waste removed and the beads are resuspended in 4-6mL of 50% Ethanol for a last wash step. Then the instrument then shakes at 500rpm for 4 minutes and subsequently captures the resin magnetically for 3 minutes.
SECTION: gDNA Isolation from supernatant
7.13. All waste is removed and 525uL low-TE Buffer is added to each tube. Samples are shaken at 500rpm for 3 minutes, then at 400rpm for 15 minutes at 80°C. Magnetic capture is performed for 4 minutes to deplete  sample of the resin, and the eluates are transferred to 2D-barcoded tubes (SBS rack).

 

SECTION: gDNA Isolation from supernatant
7.14. The instrument is finished. 
Nucleic acid concetration is determined by either NanoDrop or Quantus measurement.


 

SECTION: RNA isolation from Tempus tubes
6.9. Load an execute the protocol for "simplyRNA Blood" on the Maxwell® RSC instrument.
Running time is approx. 45min.