db-1206 diana automated rna isolation kit for agilent ...€¦ · diana automated rna isolation kit...

DIANA Biotechnologies s.r.o., Nad Safinou II 366, 25250 Vestec, Czech Republic Email: [email protected], Web: www.dianabiotech.com 1

DB-1206

DIANA Automated RNA Isolation Kit

for Agilent Bravo

User Manual

Manual Version: DB-1206-001-200707

Revision: 1.1 EN

Last update: 19th August 2020

DB-1206-10x96rxns

mailto:[email protected]

http://www.dianabiotech.com/

DIANA Automated RNA Isolation Kit for Agilent Bravo - User Manual Version DB-1206-001-200707-1.1 EN


Table of Contents 1 Preface .......................................................................................................................................................... 4

1.1 Purpose and Kit Usage .......................................................................................................................... 4 1.2 Recommended use of RNA Isolation control ........................................................................................ 4 1.3 Safety Notice ......................................................................................................................................... 5 1.4 General precautions .............................................................................................................................. 5

2 List of Materials ............................................................................................................................................ 5 2.1 Kit Components ..................................................................................................................................... 5 2.2 Required laboratory equipment ........................................................................................................... 6

2.2.1 Consumables ................................................................................................................................. 6 2.2.2 Equipment and Instruments .......................................................................................................... 7 2.2.3 Agilent Bravo liquid handling station ............................................................................................. 7

3A Before using the kit for the first time ......................................................................................................... 7 3.1 Visual inspection of the kit components ............................................................................................... 7 3.2 Reconstitution of Lysis Buffer Concentrate .......................................................................................... 7

3B Before each use of the kit .......................................................................................................................... 7 3.3 REAGENT and BEAD plates preparation ................................................................................................ 7 3.4 Preparation of the Lysis Buffer ............................................................................................................. 8 3.5 Sample Preparation............................................................................................................................... 9

3.5.1 Nasopharyngeal swabs .................................................................................................................. 9 3.5.2 Other type of biological matrix ...................................................................................................... 9

3.6 LYSIS&SAMPLE plate preparation ......................................................................................................... 9 4 Automated RNA Isolation protocol on Agilent Bravo ................................................................................. 11

4.1 Basic Instructions ................................................................................................................................ 11 4.2 Running RNA isolation protocol on Agilent Bravo .............................................................................. 11

4.2.1 Set-up Agilent Bravo station ........................................................................................................ 11 4.2.3 Run the RNA Isolation protocol at Bravo ..................................................................................... 12 4.2.4 Labware clean-up after RNA Isolation protocol .......................................................................... 13

5 Automated RT-PCR Assay plate preparation protocol ............................................................................... 15 5.1 Basic Instructions ................................................................................................................................ 15 5.2 Preparation of RT-PCR Master Mix reservoir ...................................................................................... 15 5.3 Running RT-PCR Assay plate preparation on Agilent Bravo ................................................................ 15

5.3.1 Set-up Agilent Bravo station ........................................................................................................ 15 5.3.2 Place proper labware into Agilent Bravo ..................................................................................... 16 5.3.3 Run the Assay plate preparation protocol ................................................................................... 16 5.3.4 Labware clean-up after Assay plate preparation protocol .......................................................... 17

5 Legal notice ................................................................................................................................................ 18





6 Used graphical symbols .............................................................................................................................. 19

7 FAQ ............................................................................................................................................................. 20

8 Appendix..................................................................................................................................................... 21 8.1 Quadrants layout in 384-well plate ..................................................................................................... 21 8.2 Reverse pipetting technique (manual pipettes) ................................................................................. 22





1 Preface

1.1 Purpose and Kit Usage

DIANA Automated RNA Isolation Kit is intended for isolation of RNA from biological samples using

Agilent Bravo automated liquid handling station. The kit content is sufficient for 960 isolations

(10 sets of 96 samples in 96-well plate format).

The kit contains reagents and other consumables in a format which is intended to be used by

running “DIANA Protocol for automated RNA isolation in 96-well plates” on Agilent Bravo

(described in section 4). In general, the kit can be used to isolate RNA from different types of

biological material. Isolated RNA can be then used for different types of downstream analysis,

most common being RT-PCR analysis. For this purpose, RT-PCR Master Mix reservoir is included

in the kit in order to be used together with “DIANA Automated Assay plate preparation protocol”

for Agilent Bravo to prepare the RT-PCR assay plates at Bravo. The application of this protocol is

described in this manual in section 5. Both protocols, for Automated RNA Isolation and Assay plate

preparation, are part of Installation package for automated RNA Isolation (DB-1214-Install-96).

Specific application of the kit is to isolate the RNA of SARS-CoV-2 virus from clinical samples. In

this application, the kit is typically used in a workflow, where eluted RNA is then analyzed for

presence of SARS-CoV-2 viral RNA by RT-PCR. DIANA RT-PCR kit for multiplexed SARS-Cov-2

detection (cat. no. DB-1211) is the recommended option for such combination with this kit.

This kit has been validated for isolation of SARS-CoV-2 viral RNA from phosphate buffered saline

or from Copan Universal Transport Media, both clean or with addition of diluted human sputum

(10%), saliva (10%) or blood (1%). Moreover, it has been validated for isolation of RNA from intact

cultivated SARS-Cov-2 virus in DMEM medium with 2% fetal bovine serum. It has been also

validated for isolation of RNA from SARS-Cov-2 virus in clinical samples: (i) human nasopharyngeal

swabs collected to viral transport medium and (ii) human saliva. However, it is the sole

responsibility of the end user to validate the isolation efficiency for his/her particular purpose

using positive and negative controls.

This kit is designed for research use only and not for in vitro diagnostics (IVD). If used within IVD

diagnostic workflow together with CE IVD marked RT-PCR diagnostic kits, it is the sole

responsibility of the user to validate the RNA isolation process.

1.2 Recommended use of RNA Isolation control

To validate successful RNA isolation in downstream RT-PCR applications, it is recommended to

use an RNA isolation control. The control RNA needs to be added to the Lysis Buffer to monitor

efficient purification of each sample. The control RNA is not part of the kit and must be provided

separately according to the specific application (e.g. it is part of DIANA RT-PCR kit for multiplexed

SARS-Cov-2 detection, cat. no. DB-1211).





Use of RNA isolation control added to each sample is highly recommended. If such control is not

available, at least one negative and one positive control sample should be included in each run to

monitor overall performance of the RNA Isolation kit. The negative control should be of the same

composition as the analyzed samples. The positive control can be prepared by spiking SARS-CoV-

2 viral RNA of known concentration to the negative control sample.

1.3 Safety Notice

The kit is designed for professional use only. The material safety data sheet (MSDS) will be

provided on request. Follow the general principles of chemical safety. Wear protective equipment

(gloves, goggles) and avoid contact with chemicals. Refrain from eating or drinking in laboratory

areas. Dispose the waste in designated containers and do so in accordance with legislation.

Lysis Buffer Concentrate and Wash 1 Buffer Concentrate contain Guanidium Isothiocyanate,

which is toxic. Acids must not be added to these solutions or their waste bottles! Otherwise it

could result in a formation of a toxic gas.

Bead solution, Wash buffer 2 and Elution buffer contain azide of up to 0.05%, which is toxic and

upon contact with acids produces toxic gas. Wash buffer 1 contain 20% ethanol, wash buffer 2

contain 80% ethanol, which is flammable.

If you work with biological samples, pay attention to the safety rules for working with infectious

biological material, use appropriate protective equipment (shield, respirator) and work with

samples only in designated biohazard boxes or designated areas. Work with infectious samples in

BSL2 + or BSL3 laboratories. Dispose of infectious waste in accordance with the applicable

legislation.

Continuously check that the work area is free of spilled solutions, chemicals, and biological

samples. In the event of a spill, decontaminate the work area immediately.

1.4 General precautions

Good laboratory practice to avoid sample cross-contamination should be followed, including the

use of disposable filtered pipetting tips. Handling of samples and positive controls should be

spatially separated from handling of the stock solutions in the kit to avoid their contamination.

For the same reason, do not pipette solutions directly from storage bottles, pour the required

volume into a suitable container beforehand, and do not return solutions back to storage bottles.

2 List of Materials

2.1 Kit Components

Components Scale Storage

conditions Box

number [5]





Lysis Buffer Concentrate [ 2] 95 mL 18-30°C[1] 1

REAGENT plate [2] 10 pieces 18-30°C[1] 1

ELUTION plate[6] 10 pieces 18-30°C 1

WASTE plate[6] 10 pieces 18-30°C 1

LYSIS&SAMPLE plate[6] 10 pieces 18-30°C 1

RT-PCR Master Mix reservoir[6] 1 piece 18-30°C 1

Lysis buffer 50 mL tube[6] 10 pieces 18-30°C 1

Lysis buffer 25 mL reservoir[6] 10 pieces 18-30°C 1

BEAD plate [2] 10 pieces 2-8°C[3] 2

Lysis Enhancer (red cap) 2x 609 µL -20°C[4] 3

RNA Carrier (blue cap) 4x 290 µL -20°C[4] 3

DTT Concentrate (yellow cap) 2x 544 µL -20°C[4] 3

[1] – Store in dark compartment [2] – Before use - read section 3A “Before Using the kit for the first time” [3] – Do not Freeze (frozen beads cannot be used) [4] – Avoid repeated freeze-thaw cycles (if you intend to use the component vials repeatedly, divide them into single use aliquots during their first use and store at -20°C upon use). [5] – Kit is divided into 3 boxes for shipping purposes [6] – Empty plastic provided with the kit

2.2 Required laboratory equipment

2.2.1 Consumables

• Gloves and other protective equipment

• 3 standard plastic cover lids to cover LYSIS&SAMPLE plate, ELUTION plate and RT-PCR Master

Mix reservoir during the protocols (lids can be washed and reused)

• Disposable pipette tips - 10 µL, 200 µL and 1000 µL (filter tips recommended)

• 100% Isopropanol

• Agilent Bravo 30 µL tips – box of 384 (catalogue number 11484-302) – full tip box will be

consumed; half will be used for RNA Isolation from 96 samples and other half for Assay plate

preparation

• Agilent Bravo 70 µL tips – box of 384 (catalogue number 19133-202) – full tip box will be

consumed for RNA Isolation from 96 samples

• Empty original Agilent Bravo tip boxes (part of Installation package for automated RNA

Isolation (cat No DB-1214))

• Optional: sealing foils to seal the RNA elustion plate for storage

• Optional – only required for downstream RT-PCR analysis of isolated RNA: 96-well PCR plate[1]

suitable for your qPCR instrument (RT-PCR Assay plate) and corresponding optical seal.





[1] – provided protocol for Agilent Bravo for preparation of the Assay plate and mixing the eluted RNA with

qPCR Master Mix is validated for 4ti-0951 plate (4ti-tude) and will work for any plate with identical well

geometry. For plates with other geometry, the protocol must be validated and may need to be adjusted.

2.2.2 Equipment and Instruments

• Handheld single-channel (200 µL and 1000 µL) and multichannel (200 µL) pipettes

• Benchtop centrifuge for standard SBS microplates

• Benchtop centrifuge for 1.5/2.0 mL tubes

• Test tube shaker/vortex

• Recommended: automated plate sealer

2.2.3 Agilent Bravo liquid handling station This kit is designed to be used on Agilent Bravo liquid handling station. The provided manual

expects from the user the basic knowledge of how to operate Agilent Bravo station. The manuals

in PDF forms for Agilent Bravo are provided together with Installation package for automated RNA

Isolation (DB-1214). We advise each user to get familiar with at least the Quick Guide Manual

(G5409-90020A_EN).

3A Before using the kit for the first time

3.1 Visual inspection of the kit components

Visually inspect all components of the kit for the sign of damage or leak. Do not use any kit

component which is damaged (e.g., leaked bottle, plate with peeled or pierced foil etc.).

3.2 Reconstitution of Lysis Buffer Concentrate

1. The “Lysis Buffer Concentrate” can form precipitate during shipment or storage below 18°C.

Therefore, please inspect the bottle for visible precipitation before first use.

2. If precipitate is present – place bottle into thermal incubator set to 37 °C for one hour and

mix afterwards by turning bottle upside down several times – precipitate should dissolve,

and stock solution should not precipitate if stored at room temperature.

Note: for storage at temperatures at or below 18°C, we recommend inspecting bottle for

precipitate before every use and reconstituting the concentrate as described above if the

precipitate is present.

3B Before each use of the kit

3.3 REAGENT and BEAD plates preparation

1. Spin BEAD plate at 200g for 1 min before use

2. Spin REAGENT plate at 200g for 1 min before use





Note: Use proper balance plates for both BEAD and REAGENT plate. Do not spin only these two plates since they have

different weight.

3.4 Preparation of the Lysis Buffer

1. The Lysis Buffer should be always prepared fresh in the amount required for immediate use.

2. Components Lysis Enhancer, RNA Carrier and DTT Concentrate are frozen and need to be

thawed completely before use, until clear solution is formed. To ensure homogeneity of

thawed stock solutions always spin down all fluids to the vial bottom, vortex and spin down

once again before pipetting the solution from each vial.

3. Upon thawing keep RNA Carrier and Lysis Enhancer on ice.

4. Prepare the Lysis Buffer according to the table 3.4 (the table already accounts for the dead

volume needed for Lysis buffer handling). The table outlines the preparation of the Lysis

buffer for the isolation of RNA from 96 samples using one LYSIS&SAMPLE plate. For isolation

of more samples adjust the volumes based on the number of LYSIS&SAMPLE plates used.

Table 3.4: List of reagents needed for preparation of Lysis buffer

Order of Addition

Components Sample Volume [µL]

1 plate (96 samples)

1 Lysis Buffer Concentrate 8100

2 Lysis Enhancer [1] 113

3 RNA Carrier [1] 108

4 DTT concentrate 102

5 RNA isolation control [1,2] 128[3]

6 100 % Isopropanol [2] 7410

Total volume 15961

[1] – Keep on ice after thawing [2] - Not included in the kit [3] – If not using RNA isolation control, add the same volume of water instead.

5. All the components (1-6) should be pipetted in the same order as indicated in the table above

into the provided 50 mL tube and mixed thoroughly. If Lysis buffer for 2 plates is prepared,

single 50 mL falcon tube can be used for preparation. However, if Lysis buffer for more than

two plates is prepared at once use more 50 mL falcon tubes accordingly. Resulting solution

containing all the components 1 to 6 is referred in text as the "Lysis Buffer".

6. Pour prepared Lysis buffer into provided plastic reservoir (capacity 25 mL). Dispense 140 µL

of Lysis buffer into the bottom of each well of LYSIS&SAMPLE plate using multichannel

pipette.





IMPORTANT: Dispense of Lysis buffer into LYSIS&SAMPLE plate should be done quickly (ideally

within 1-2 minutes after pouring the buffer into the reservoir) to limit potential evaporation of the

Lysis buffer from the reservoir. If the last aspiration of Lysis buffer by multichannel pipette is not

possible due to not enough volume, fill the rest of the plate by single-channel pipette. Optionally,

electronic 1250 µL multichannel pipette or similar type of dispenser can be used to dispense Lysis

buffer into LYSIS&SAMPLE plate right from the 50 mL tube, where the Lysis buffer evaporates much

more slowly than in the reservoir.

7. Cover the LYSIS&SAMPLE plate with plastic lid to prevent contamination and evaporation.

8. If the LYSIS&SAMPLE plate will be standing for more than 30 minutes before addition of

analyzed samples, seal the LYSIS&SAMPLE plate with non-permeable foil.

Note: Sealed LYSIS&SAMPLE plate can be used up to 8 hours from preparation time if stored in dark compartment at

room temperature. Large batch of Lysis Buffer can be conveniently prepared in the morning of experiment day and used

throughout the day.

3.5 Sample Preparation

Prior addition to the lysis buffer, all samples must be treated as potentially infectious material

and must be handled accordingly.

3.5.1 Nasopharyngeal swabs For RNA isolation from a nasopharyngeal swab in virus transport medium vortex the swab in a

sealed vial for 1 minute to release biological material and then spin down briefly.

3.5.2 Other type of biological matrix For RNA isolation from other types of biological samples apply standard sample preparation steps

accordingly.

Optional: To reduce infectivity the sample can be heat-inactivated for 10 minutes at 65°C.

However, possible impacts of the heat inactivation on the RNA recovery must be evaluated for

each analyzed RNA type and biological matrix.

3.6 LYSIS&SAMPLE plate preparation

1. Remove the lid (or peel out the seal) from the LYSIS&SAMPLE plate.

2. Dispense 60 µL of sample into appropriate well. The sample should be dispensed straight into

the lysis buffer NOT on the side of well wall. Mix once to wash out the sample from the tip.

Do not over-dispense during mixing to minimize formation of bubbles within the mixture.

Additional mixing is not needed since it will be done during RNA isolation protocol by Bravo.

IMPORTANT: During sample dispense into LYSIS&SAMPLE plate, it is recommended to cover the

plate with plastic lid to minimize the evaporation between addition of each sample.

Cover the prepared LYSIS&SAMPLE plate with plastic lid and transfer it into the Agilent Bravo

station.





3. The total time of handling of the opened LYSIS&SAMPLE plate (either after peeling the foil or

after preparation) must not exceed 2 hours, or up to 4 hours if the plate is covered with a lid

which is removed only for pipetting. The plate can be stored sealed with impermeable foil,

however the total time since preparation of the Lysis buffer (including the time of adding the

samples) until the Bravo protocol is started must not exceed 8 hours.





4 Automated RNA Isolation protocol on Agilent Bravo

4.1 Basic Instructions

1. Make sure that Agilent Bravo station is properly installed and tested and automated RNA

isolation protocol files have been installed. The recommended installation and testing

procedures can be found in manual for Installation package for automated RNA isolation in

96-well plates on Agilent Bravo (DB-1214-Install-96).

2. Run RNA isolation at room temperature (approx. 18-25°C).

3. The following procedure performs simultaneous RNA Isolation from 96 samples yielding 18 µL

of eluted RNA in quadrant 1 (Q1) of the ELUTION plate (see Appendix 1 for description of

quadrants layout in 384-well plates).

4.2 Running RNA isolation protocol on Agilent Bravo

4.2.1 Set-up Agilent Bravo station 1. Start VWorks program and log in.

2. Switch ON the Agilent Bravo

3. Open Bravo device file named Bravo3 96ST_magnet_pos5.

4. Initialize Bravo station along the manufacture’s manual.

5. Open Bravo protocol file named DIANA_RNA_isol_DB-1206_v1.3.

6. The VWForms window (screenshot shown below) will appear automatically.

Figure 4.2.1: Screenshot of VWForms window of RNA Isolation protocol

7. Fill out the fields “Experiment ID”, “Operator Name” and “Elution plate ID” (the Elution plate

ID can be found on the side of the ELUTION plate and is unique for each plate)





4.2.2 Place proper labware into Agilent Bravo It is more convenient to start loading the Bravo station with labware from back positions (1/2/3)

to front positions (7/8/9). Try to avoid passing a labware above opened tip boxes or plates.

Next steps should be done with clean gloves to avoid any contamination of the plates and the

RNA isolation protocol must be started as soon as possible after peeling the foils from the plates

(start with these steps only after you have prepared the LYSIS&SAMPLE plate).

1. Proper “Labware Position Layout” in the Agilent Bravo station is shown on the scheme in the

VWForms window (the orientation of Bravo station is such that positions 7,8 and 9 are the

user-facing front positions).

2. Place empty tip box, WASTE plate (peel off the seal beforehand) and full tip70 tip box into

Bravo station at positions 1,2, and 3, respectively. Remove the lid from 70 µL tip70 tip box

and empty tip box.

3. Outside of Bravo station, peel off the seal from BEAD plate carefully. It is important that no

liquid or beads are splashed during the peeling. Place BEAD plate at position 5 on Magnetic

adapter in Bravo station.

4. Outside of Bravo station, peel off the seal from REAGENT plate carefully. It is important that

no liquid are splashed during the peeling. Place REAGENT plate at position 6 in Bravo station.

5. Place full 30 µL tip30 tip box into Bravo station at positions 9 and remove its lid.

Note: If Automated Assay plate preparation protocol is used, then the whole tip30 tip box is consumed. If not, then after

the RNA Isolation protocol is finished the box will still contain clean unused 30 µL tips in quadrants 3 and 4. Such tip box

can be used for one more RNA Isolation protocol, but the box MUST be turned by 180° before next use!!!

6. Peel off the ELUTION plate and place into Bravo station at position 7.

7. As a last step, place the lidded LYSIS&SAMPLE plate into Bravo station at position 8 and then

remove the lid from the plate.

4.2.3 Run the RNA Isolation protocol at Bravo Run the protocol according to the protocol instructions. Details may differ according to the

protocol version, but typically requiring following set of commands:

Before starting a run, carefully check:

• All plates, tip boxes and magnet are in the right position (Photo of labware distribution

in Agilent Bravo station for RNA Isolation is shown in section 7.3)

• Front side of each item faces toward you

• The number on each item corresponds to the number of the position

• Each item sits precisely in its designated position

1. Press the “Run” button and start the RNA Isolation protocol on Bravo.

2. After clicking “Run” new windows appears on the screen providing more specific of the

protocol and tips usage. Read and follow the instructions. Click “Finish” on that window.

3. New message appears that summarizes the layout of each labware on its designated position.

Check that the layout corresponds to the physical layout of your experiment.

4. Check again that all lids are removed, and all seals are peeled off.





5. Click “Continue” button that starts the RNA Isolation protocol.

6. During the RNA Isolation protocol following steps occur: i) the LYSIS&SAMPLE plate is mixed,

ii) solution is transferred to the BEAD plate, mix with magnetic beads and incubated,

iii) solution is aspirated from beads on magnetic adapter, iv) beads are sequentially washed

three times with washing solutions, v) elution buffer is added to the beads and incubated,

iv) elution buffer is aspirated from the beads on the magnetic adapter and transferred to the

ELUTION plate.

7. The protocol typically takes 40 minutes to complete and does not require any intervention of

the operator during this time.

Note: We recommend the user to visually check the RNA Isolation protocol for two or three initial runs upon installation

to make sure that all liquid handling tasks are performed correctly.

8. After protocol finishes click “Export PDF report” button to create PDF report of the protocol

that will include name of the used Bravo protocol, time, operator name, experiment ID and

elution plate ID.

4.2.4 Labware clean-up after RNA Isolation protocol After protocol is finished 18 µL of eluted RNA from 96 samples is present in the ELUTION plate in

a quadrant 1 (wells A1, A3, A5, etc.; see section 7.1).

IMPORTANT: Commonly 5 µL of eluted RNA is used for RT-PCR analysis (volume depends on used

RT-PCR kit). Since 18 µL of eluted RNA has been yielded from RNA Isolation, up to three RT-PCR

reactions can be set from the one RNA isolation. For long-term storage of eluted RNA, we

recommend sealing the ELUTION plate with freezing-compatible foil and store at -80°C.

4.2.4.1 Clean-up before RT-PCR Assay plate preparation protocol 1. Lid the elution plate with clean plastic lid to prevent any potential contamination.

2. Lidded ELUTION plate (pos 7), tip30 tip box (pos 9) and tip70 tip box (pos 3) stay within Bravo

station at the same positions for DIANA Assay plate preparation protocol.

3. Discard the LYSIS&SAMPLE, BEAD, WASTE, and REAGENT plate.

IMPORTANT: LYSIS&SAMPLE, BEAD and WASTE plates can potentially contain viral RNA and

therefore must be disposed in a way that minimizes the risk of contamination of the working area.

If possible, we recommend sealing these plates after the RNA Isolation protocol is finished and

then dispose them accordingly.

4. Lid and discard whole “empty tip box” (pos 1), which contains used 70 µL tips.

IMPORTANT: Used tips may contain viral RNA and thus must be disposed accordingly.

5. Close VWForms window and protocol window in VWorks program.

4.2.4.2 Clean-up ONLY in case RT-PCR Assay plate is NOT prepared in Bravo 1. Lid the elution plate with clean plastic lid to prevent any potential contamination.

2. Take out lidded ELUTION plate (pos 6) from Bravo station.

3. If full “tip30 tip box” was used for RNA Isolation protocol, lid it and keep it.





Note: The tip box now contains clean unused 30 µL tips in Q3 and Q4. Such tip box can be used for one more RNA

Isolation protocol, but it MUST be turned by 180° before the next use so the clean tips are in Q1 and Q2 (see section 7.1).

4. Discard the LYSIS&SAMPLE, BEAD, WASTE, and REAGENT plate.

IMPORTANT: LYSIS&SAMPLE, BEAD and WASTE plates can potentially contain viral RNA and

therefore must be disposed in a way that minimizes the risk of contamination of the working area.

If possible, we recommend sealing these plates after the RNA Isolation protocol is finished and

then dispose them accordingly.

5. Lid and discard whole “empty tip box” (pos 1), which contains used 70 µL tips.

6. Discard used 30 µL tips from “tip70 tip box” and keep the empty tip box for next RNA Isolation

protocol as an “empty tip box”.


7. Close VWForms, protocol and device file windows in VWorks program. Close the VWorks

program and switch OFF the Bravo station.





5 Automated RT-PCR Assay plate preparation protocol This is the optional protocol which can be run subsequently to the RNA Isolation protocol

(section 4) in order to transfer eluted RNA to the Assay plate for RT-PCR analysis. The Assay plate

preparation protocol will dispense 13.5 µL of RT-PCR master mix into Assay plate (96-well format

PCR plate) and then mixes 4.5 µL of eluted RNA obtained for RNA Isolation protocol with prepared

RT-PCR master mix.

Note: The overall reaction volume of RT-PCR reaction is 18 µL (not 20 µL as commonly used in manual setups) – this is

to accommodate for a small increase of dead volumes in automated protocol without increasing consumption of RT-

PCR kit reagents.

5.1 Basic Instructions

1. Make sure that Agilent Bravo station is properly installed and tested and automated RT-PCR

assay plate preparation protocol files have been installed. The recommended installation and

testing procedures can be found in manual for product DB-1214-Install-96.

2. Run RNA isolation at room temperature (approx. 18-25°C).

5.2 Preparation of RT-PCR Master Mix reservoir

1. Follow the instructions provided by the manufacturer of the RT-PCR kit to prepare 1.6 mL of

RT-PCR master mix (this accounts for certain dead volume in subsequent pipetting steps and

is typically the volume which can be prepared from 100 sample RT-PCR kit). Prepare the RT-

PCR master mix along manufacturer instructions in a concentration that corresponds to the

RT-PCR reaction where 5 µL of sample is mixed with 15 µL of RT-PCR master mix.

DIANA RT-PCR kit for multiplexed SARS-Cov-2 detection, cat. no. DB-1211, is recommended

option for SARS-Cov-2 RNA detection in combination with this RNA Isolation kit.

2. Peel off the foil from one column (8 wells) of RT-PCR Master Mix reservoir.

3. Dispense 180 µL of RT-PCR Master Mix into each of 8 wells of RT-PCR Master Mix reservoir

using 200 µL single-channel pipette. To ensure precise pipetting, use reverse pipetting

technique (see section 7.2).

4. Cover the prepared RT-PCR Master Mix reservoir with clean plastic lid.

5. Check that there are no bubbles at the bottom of the wells of RT-PCR Master Mix reservoir.

5.3 Running RT-PCR Assay plate preparation on Agilent Bravo

5.3.1 Set-up Agilent Bravo station 1. Start VWorks program and log in (skip if run after RNA Isolation protocol)

2. Switch ON the Agilent Bravo station (skip if run after RNA Isolation protocol)

3. Open Bravo device file named Bravo3_96ST_magnet_pos5.

4. If needed initialize Bravo station along the manufacture’s manual.

5. Open Bravo protocol file named DIANA_Assay_plate_prep_DB-1206_v1.2.

6. The VWForms window (screenshot shown below) will appear automatically





Figure 5.3.1: Screenshot of VWForms window of Assay plate preparation protocol

7. Fill the Column number in RT-PCR Master Mix reservoir box (top right corner of the screen).

The value in this field must be a number in range of 1-12 and it corresponds to the column

number in RT-PCR Master Mix reservoir plate (column 1 being on the left side and column 12

on the right side of the reservoir). The column number can be found on upper side of the

reservoir. The column number must be filed correctly, otherwise the protocol will not work.

8. Fill out the fields “Experiment ID”, “Operator Name” and “Elution plate ID” (the Elution plate

ID can be found on the side of the ELUTION plate and is unique for each plate)

5.3.2 Place proper labware into Agilent Bravo It is more convenient to start loading the Bravo station with labware from back positions (1/2/3)

to front positions (7/8/9). Try to avoid passing a labware above opened tip boxes or plates.

1. From RNA Isolation protocol, the lidded ELUTION plate, “tip30 tip box” (with 30 µL clean tips

in Q3, Q4) and “tip70 tip box” (with 30 µL used tips in Q1, Q2 (see section 7.1)), are already

present at positions 7, 9 and 3, respectively.

Note: If Assay plate preparation protocol is not run right after RNA Isolation protocol, then first place the ELUTION plate,

“tip30 tip box” and “tip70 tip box” into their respective positions and remove the lids from tip boxes.

2. Place lidded RT-PCR Master Mix reservoir into Bravo station at position 2 and remove the lid.

3. Place Assay plate into metal PCR plate insert (cat.no. G5498B#G013) and then into Bravo

station at position 8

4. Remove the lid or peel off the seal from ELUTION plate.

IMPORTANT: Peeling off the seal from ELUTION plate should be done outside of Bravo station.

Peel the seal carefully. It is important that no liquid is splashed during the peeling.

5.3.3 Run the Assay plate preparation protocol Run the protocol according to the protocol instructions. Details may differ according to the

protocol version, but typically requiring following set of commands:





1. Press the “Run” button and start the RNA Isolation protocol on Bravo.

2. After clicking “Run” new windows appears on the screen providing more specific of the

protocol and tips usage. Read and follow the instructions. Click “Finish” on that window.

3. New message appears that summarizes the layout of each labware on its designated position.

Check that the layout corresponds to the physical layout of your experiment.

4. Check again that all lids are removed, and all seals are peeled off.

5. Additional message appears that ask you to re-check that correct column number of RT-PCR

Master Mix reservoir was provided.

6. Click “Continue” button that starts the Assay plate preparation protocol.

7. The protocol run takes approximately 5 minutes to complete and does not require any

intervention of the operator during this time.

Note: We recommend the user to visually check the RNA Isolation protocol for several (2-3) initial runs upon installation

to make sure that all liquid handling tasks are performed correctly.

8. After protocol finishes click “Export PDF report” button to create PDF report of the protocol

that will include name of the used Bravo protocol, time, operator name, experiment ID,

elution plate ID, column number in RT-PCR Master Mix reservoir.

5.3.4 Labware clean-up after Assay plate preparation protocol 1. After protocol finishes seal the Assay plate with optically transparent foil. The Assay plate is

now ready to be measured in the qPCR instrument.

2. Seal the ELUTION plate by freezing-resistant foil and keep it at -20°C for storage for up to

30 days or at -80°C for long-term storage. If the ELUTION plate containing the remaining RNA

is not be stored it can be disposed identically as REAGENT, BEAD and WASTE plates (see

section 4.2.4)

3. Discard the remaining unused tips from tip30 tip box and keep the tip box as new “empty tip

box” for next RNA Isolation protocol.

4. Lid and discard the tip70 tip box.


5. Lid the RT-PCR Master Mix reservoir plate. Keep the used column unsealed so it is clear which

columns in the reservoir are still ready to be used in the next Assay plate preparation protocol.

6. Close VWForms, protocol and device file windows in VWorks program. Close the VWorks

program and switch OFF the Bravo station.





5 Legal notice To the fullest extent permitted by the laws of your jurisdiction, the DIANA Biotechnologies, s.r.o.

is excluded of any liability for any direct or indirect damages associated with and / or incurred in

relation to this document and its use, as well as for any direct and indirect damages associated

with and / or incurred in relation to the DIANA Automated RNA Isolation Kit for Agilent Bravo and

its use.

The DIANA Automated RNA Isolation Kit for Bravo is intended for research use only and is not

certified for clinical diagnostics. If used within IVD diagnostic workflow together with CE IVD

marked RT-PCR diagnostic kits, it is the sole responsibility of the user to validate the RNA isolation

process.





6 Used graphical symbols Manufacturer / Výrobce

Date of Manufacture / Datum výroby

Caution / Pozor

Lot Number / Kód dávky

Operator´s manual, operating instructions / Čtěte návod k použití

Catalogue Number / Katalogové číslo

Lower limit of temperature / Dolní mez teploty

Temperature limit / Limit teploty

Upper limit of temperature / Horní mez teploty

Do not use if package is damaged / Nepoužívat jestliže je balení poškozeno

Use by date / Použít do data

Do not reuse / Nepoužívat opětovně

Keep away from sunlight / Chraňte před slunečním zářením





7 FAQ What if beads are present in the tips during liquid aspiration?

Small amounts of beads can be present in the tips during aspiration of lysis and sample or wash

solution. This has no effect on overall performance of RNA Isolation protocol. On the other hand,

if beads are present inside tip that are used for transfer of eluted RNA into ELUTION plate, then

the teaching positions of the Agilent Bravo station must be checked. Presence of beads in eluted

RNA will affect RNA Isolation protocol dramatically and protocol cannot be run until this error is

corrected.





8 Appendix

8.1 Quadrants layout in 384-well plate

Each 384-well plate can be divided into four sets of 96-wells, which correspond to four 96-well

plates. The wells corresponding to one of these 96-well plates forms one quadrant of the 384-

well plate. The scheme bellow illustrates the layout of wells in 384-well plate highlighting wells

along their quadrant in comparison with their well positions:

• Quadrant 1 (Q1) colored blue correspond to wells A1, A3, A5, ……, O21, O23

• Quadrant 2 (Q2) colored green correspond to wells A2, A4, A6, ……, O21, O24

• Quadrant 3 (Q3) colored red correspond to wells B1, B3, B5, ……, P21, P23

• Quadrant 4 (Q4) colored white correspond to wells B2, B4, B6, ……, P22, P24

Figure 7.1.1: Scheme of quadrants layout in 384-well format plate

Figure 7.1.2: Scheme of well positions in 384-well format plate

384wp 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24

A 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2

B 3 4 3 4 3 4 3 4 3 4 3 4 3 4 3 4 3 4 3 4 3 4 3 4

C 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2

D 3 4 3 4 3 4 3 4 3 4 3 4 3 4 3 4 3 4 3 4 3 4 3 4

E 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2

F 3 4 3 4 3 4 3 4 3 4 3 4 3 4 3 4 3 4 3 4 3 4 3 4

G 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2

H 3 4 3 4 3 4 3 4 3 4 3 4 3 4 3 4 3 4 3 4 3 4 3 4

I 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2

J 3 4 3 4 3 4 3 4 3 4 3 4 3 4 3 4 3 4 3 4 3 4 3 4

K 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2

L 3 4 3 4 3 4 3 4 3 4 3 4 3 4 3 4 3 4 3 4 3 4 3 4

M 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2

N 3 4 3 4 3 4 3 4 3 4 3 4 3 4 3 4 3 4 3 4 3 4 3 4

O 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2

P 3 4 3 4 3 4 3 4 3 4 3 4 3 4 3 4 3 4 3 4 3 4 3 4

384wp 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24

A A01 A02 A03 A04 A05 A06 A07 A08 A09 A10 A11 A12 A13 A14 A15 A16 A17 A18 A19 A20 A21 A22 A23 A24

B B01 B02 B03 B04 B05 B06 B07 B08 B09 B10 B11 B12 B13 B14 B15 B16 B17 B18 B19 B20 B21 B22 B23 B24

C C01 C02 C03 C04 C05 C06 C07 C08 C09 C10 C11 C12 C13 C14 C15 C16 C17 C18 C19 C20 C21 C22 C23 C24

D D01 D02 D03 D04 D05 D06 D07 D08 D09 D10 D11 D12 D13 D14 D15 D16 D17 D18 D19 D20 D21 D22 D23 D24

E E01 E02 E03 E04 E05 E06 E07 E08 E09 E10 E11 E12 E13 E14 E15 E16 E17 E18 E19 E20 E21 E22 E23 E24

F F01 F02 F03 F04 F05 F06 F07 F08 F09 F10 F11 F12 F13 F14 F15 F16 F17 F18 F19 F20 F21 F22 F23 F24

G G01 G02 G03 G04 G05 G06 G07 G08 G09 G10 G11 G12 G13 G14 G15 G16 G17 G18 G19 G20 G21 G22 G23 G24

H H01 H02 H03 H04 H05 H06 H07 H08 H09 H10 H11 H12 H13 H14 H15 H16 H17 H18 H19 H20 H21 H22 H23 H24

I I01 I02 I03 I04 I05 I06 I07 I08 I09 I10 I11 I12 I13 I14 I15 I16 I17 I18 I19 I20 I21 I22 I23 I24

J J01 J02 J03 J04 J05 J06 J07 J08 J09 J10 J11 J12 J13 J14 J15 J16 J17 J18 J19 J20 J21 J22 J23 J24

K K01 K02 K03 K04 K05 K06 K07 K08 K09 K10 K11 K12 K13 K14 K15 K16 K17 K18 K19 K20 K21 K22 K23 K24

L L01 L02 L03 L04 L05 L06 L07 L08 L09 L10 L11 L12 L13 L14 L15 L16 L17 L18 L19 L20 L21 L22 L23 L24

M M01 M02 M03 M04 M05 M06 M07 M08 M09 M10 M11 M12 M13 M14 M15 M16 M17 M18 M19 M20 M21 M22 M23 M24

N N01 N02 N03 N04 N05 N06 N07 N08 N09 N10 N11 N12 N13 N14 N15 N16 N17 N18 N19 N20 N21 N22 N23 N24

O O01 O02 O03 O04 O05 O06 O07 O08 O09 O10 O11 O12 O13 O14 O15 O16 O17 O18 O19 O20 O21 O22 O23 O24

P P01 P02 P03 P04 P05 P06 P07 P08 P09 P10 P11 P12 P13 P14 P15 P16 P17 P18 P19 P20 P21 P22 P23 P24





8.2 Reverse pipetting technique (manual pipettes)

Reverse pipetting is a technique to dispense a measured quantity of liquid by means of air

displacement, which reduces the risk of splashing, foam or bubble formation. Reverse pipetting is

more precise in dispensing small volumes of liquids containing proteins and biological solutions

compared to forward pipetting.

Reverse pipetting – step-by-step

Figure 7.2.1: Scheme of reverse pipetting technique

1. Attach tips to the handheld single/multi-channel pipette and make sure all tips are tight.

2. Press the piston past the first stop (if filter tips are used and dispensed liquid is close the

maximum capacity of the tip, make sure not to aspirate the liquid into the filter)

3. Dip the tip into the solution to a certain depth, according to the set volume, and slowly release

the piston. This action will fill the tip with a volume that is larger than the set volume.

4. Withdraw the tip from the liquid and dispense the liquid into the receiving vessel by pressing

the operating button gently and steadily to the first stop. This volume is equal to the set

volume. Hold the button in this position. Some liquid will remain in the tip, and this liquid

should not be dispensed.

5. Continue pipetting by repeating steps 3 and 4.

6. After all wells have been filled with the liquid dispense the left-over volume from the tip back

into the reservoir (step X).

7. Discard the tip

For electronic pipettes, the reverse pipetting technique is also preferred way and equals to setting

the dead volume (preaspirate) to a nonzero volume.



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