PRRS NA/HP detection kit Introduction

Instruction Manual

Reverse transcriptase Polymerase Chain Reaction (RT-PCR) in real time for the qualitative detection of

North America Porcine Reproductive and Respiratory Syndrome virus

KIT HQS_PRRS-NA_i_tqm with IPC

48/96 reactions

The kit include and internal positive control

The kit uses the Taqman® probe technology to measure DNA/RNA amplification

Storage conditions:

Keep all reagents at -20 °C until use and after the first use.

Manufacturer
ADNUCLEIS SAS
Siret 497 802 165 000 17
TVA intraCEE : FR 3649780216500017
* 3 Route des Pierres Blanches
F- 69290 GREZIEU LA VARENNE
FRANCE

RT-PCR kit content-storage

rev 1.0 (13/07/2016)

IV.               Description and content of the kit

The RT-PCR kit is ready to use for the specific detection of a target. The kit contains reagents and enzymes necessary of the amplification of a specific target, synthetic Target positif RNA (positive control) and of RNA inhibitory control (IPC) (see table 1). Fluorescence is emitted and measured individually by an optical system during PCR. The detection of the amplified fragment is usually carried out by the fluorimeter using the channels listed in table 2.

Table 1:

Components of the kits 48 reactions 96 reactions Reconstitution
Volume (µl) Volume (µl)
PCR pre-mix
1160 2000 Ready to use
Taq polymerase Nucleis (Taq N) 116 200 Ready to use
Reverse transciptase Nucleis (RT N) 58 100 Ready to use
Target RNA Positive control 200 200 Ready to use
RNA IPC (inhibitory control) 150 300 Ready to use

Table 2: Fluorophores usually used in duplex target-IPC kits.

Target Fluorophore Excitation Emission
Target FAM 499 519
IPC VIC 520 548

(Please check the kit certificat for the fluorophore used in a specific kit.)

Alternatives:

-channel FAM: (ABI Systems, smartcycler II; Chromo 4/CFX96, System Agilent MX and aria, realplex), channel 530 (LC480), channel Green (RotorGene)

-channel VIC: Channel VIC (ABI systems, Biorad CFX96, Agilent Mx and aria, realplex), channel Alexa 532 (Smart cyclerII), Channel 560 (LC 480); channel Yellow  (RotorGene)

 

Required material and reagents not provided

  • Elution buffer
  • A RNA extraction and purification kit
  • Biological hood
  • qPCR instrument
  • Micro centrifuge
  • Vortex
  • Plates / tubes for qPCR
  • Micropipettes
  • Filtre tips for micropipettes
  • Sterile microtubes
  • A viral RNA extraction and purification kit
  • Non powdered gloves

V.               Storage

All reagents must be stored at -20°C. Storage at 4°C is forbidden.

All reagents can be used until the expiration date indicated on the kit

Many freezing/thawing cycles (>3x) must be avoided, could lead to decrease in sensitivity

VI.               Cautions and notes

  • Read carefully instructions before starting.
  • The experiment must be performed by competent staff
  • Instruments must have been properly installed, calibrated and maintained according to the manufacturer’s recommendations
  • Clinical samples are potentially infectious and must be processed under a laminar flow hood/
  • The experiment must be performed according to good laboratory practices
  • Do not use this kit after expiration date
  • Many freezing/thawing cycles of the reagents must be avoided, and could lead to decrease of sensitivity
  • Once defrosted, spin down briefly the tubes before use
  • Spin down briefly enzymes tubes to pellet the viscous content at the bottom of the tubes.
  • Use of ice or cooling block is mandatory to prepare the mix and dispense reconstituted mix in wells.
  • Define 3 working areas: 1)Isolation of DNA/RNA, 2) Preparation of the reaction mix and 3) Amplification/detection of amplified products.
  • Pipettes, reagents and other materials must not cross each area
  • Specific caution is required to preserve the purity of the reagents and reaction mixtures. The use of gloves is mandatory. Appropriate methods of preparation of DNA should be used.
  • Always use filtered tips for micropipettes
  • Use specific lab coat and gloves in each working area
  • Do not pipette with mouth and do not eat, drink or smoke in the area
  • Avoid sprays

VII.               Collection of samples, transport and storage

  • Collect samples in sterile tubes
  • The samples should be extracted immediately or frozen from -20°C to 80°C
  • Transport of clinical samples must obey local regulations for this type of infectious agents.

real Time RT-PCR principles-taqman

III.               Principle of the detection

The ADNUCLEIS RT-PCR kits are using reverse transcription to synthesize RNA into cDNA followed by the amplification of the cDNA by real time RT- PCR. Kits may also include  inhibition control (IPC) made of synthetic RNA. This control ensures that a negative result may not be due to the presence of PCR inhibitors at high quantity.

The test is performed from the RNA extracted from the sample. For taqman duplex kits with IPC, the target is usually detected by  the FAM channel and the IPC by the JOE/VIC channel (color Probe may be different, please refer to the kit certificat for further information). All emit at a specific fluorescence following its hydrolysis during the elongation of the amplification product. The measurement of the intensities of the real time fluorescence relates the accumulation of specific amplification products.

This amplification system has been validated on:

  • A synthetic RNA produced in vitro from a plasmid containing a specific insert of the target or of the IPC.

IV.               Description and content of the kit

The PRRS NA virus real time RT-PCR kit is ready to use for the specific detection of the type 2 (NA/HP) PRRS virus. The kit contains reagents and enzymes necessary of the amplification of type 2 (NA/HP) PRRS virus, synthetic PRRS NA RNA (positive control) and of RNA control (IPC) of RT-PCR inhibition (see table 1). Fluorescence is emitted and measured individually by an optical system during PCR. The detection of the amplified fragment is carried out by the fluorimeter using the channels listed in table 2.

 

Table 1:

Components of the kits 48 reactions 96 reactions Reconstitution
Volume (µl) Volume (µl)
PCR pre-mix 1160 2000 Ready to use
Taq polymerase Nucleis (Taq N) 116 200 Ready to use
Reverse transciptase Nucleis (RT N) 58 100 Ready to use
PRRS NA RNA Positive control 200 200 Ready to use
RNA IPC (inhibitory control) 150 300 Ready to use

 

Table 2

Target Fluorophore Excitation Emission
PRRS NA virus FAM 499 519
IPC VIC 520 548

 

Alternatives:

-channel FAM: (ABI Systems, smartcycler II; Chromo 4/CFX96, System Agilent MX and aria, realplex), channel 530 (LC480), channel Green (RotorGene)

-channel VIC: Channel VIC (ABI systems, Biorad CFX96, Agilent Mx and aria, realplex), channel Alexa 532 (Smart cyclerII), Channel 560 (LC 480); channel Yellow  (RotorGene)

 

Required material and reagents not provided

  • Elution buffer
  • A RNA extraction and purification kit
  • Biological hood
  • qPCR instrument
  • Micro centrifuge
  • Vortex
  • Plates / tubes for qPCR
  • Micropipettes
  • Filtre tips for micropipettes
  • Sterile microtubes
  • A viral RNA extraction and purification kit
  • Non powdered gloves

V.               Storage

All reagents must be stored at -20°C. Storage at 4°C is forbidden.

All reagents can be used until the expiration date indicated on the kit

Many freezing/thawing cycles (>3x) must be avoided, could lead to decrease in sensitivity

VI.               Cautions and notes

  • Read carefully instructions before starting.
  • The experiment must be performed by competent staff
  • Instruments must have been properly installed, calibrated and maintained according to the manufacturer’s recommendations
  • Clinical samples are potentially infectious and must be processed under a laminar flow hood/
  • The experiment must be performed according to good laboratory practices
  • Do not use this kit after expiration date
  • Many freezing/thawing cycles of the reagents must be avoided, and could lead to decrease of sensitivity
  • Once defrosted, spin down briefly the tubes before use
  • Spin down briefly enzymes tubes to pellet the viscous content at the bottom of the tubes.
  • Use of ice or cooling block is mandatory to prepare the mix and dispense reconstituted mix in wells.
  • Define 3 working areas: 1)Isolation of DNA/RNA, 2) Preparation of the reaction mix and 3) Amplification/detection of amplified products.
  • Pipettes, reagents and other materials must not cross each area
  • Specific caution is required to preserve the purity of the reagents and reaction mixtures. The use of gloves is mandatory. Appropriate methods of preparation of DNA should be used.
  • Always use filtered tips for micropipettes
  • Use specific lab coat and gloves in each working area
  • Do not pipette with mouth and do not eat, drink or smoke in the area
  • Avoid sprays

VII.               Collection of samples, transport and storage

  • Collect samples in sterile tubes
  • The samples should be extracted immediately or frozen from -20°C to 80°C
  • Transport of clinical samples must obey local regulations for this type of infectious agents.

VIII.               Procedure

VIII.1   RNA/DNA extraction

RNA/DNA extraction kits are available from ADNUCLEIS.

For semen samples, the RNA extraction kit and RNA/DNA purification kit of ADNUCLEIS is recommended.

The RNA IPC on the Vic channel added to the RT-PCR reaction ensures that a negative result is not due to the presence of high amount of RT-PCR inhibitors. In that case, 3 µl of RNA IPC is added per reaction.

VIII.2   Q RT-PCR

General comment: Positive control contains a high concentration of nucleic acids. Manipulations must be performed carefully to avoid contamination.

It is necessary to test the positive control PRRS NA RNA (12 µl in final volume of 52 µl reaction), as well as a negative control (elution buffer, supplied separately). RNA IPC is added in each RT-PCR well (3µl) or in the reconstituted mix at the appropriate volume (3 µl * number of reaction prepared).

Example of qualitative real time RT-PCR plate

 

  1 2 3 4 5 6 7 8 9 10 11 12
A Positive control + IPC
B IPC
C Negative control
D Sample 1 + IPC
E Sample 2 + IPC
F
G
H

Positive control: PRRS NA synthetic RNA

IPC: synthetic RNA to control inhibition properties of the sample.

VIII.3   Real time RT-PCR protocol

The Reverse transcriptase reaction is performed before the PCR. The Taq N polymerase is added after the reverse transcriptase reaction is performed.

 

  • Homogenize gently the premix tube before starting and spin down brief the premix tubes, enzymes tubes and control tubes.
  • Prepare the reaction mix as follows by multiplying the number of N samples to test (including positive, IPC, negative controls). On average, prepare enough reagents for N+3 reactions minimum.
Number of reactions (N) 1 N+3
Premix 18 µl (N+3)x 18 µl
RT N 1 µl (N+3)x 1 µl
IPC 3 µl (N+3)x 3 µl
Elution buffer 12 µl (N+3)x 12 µl
Total volume of Reaction mix 34 µl (N+3)x 34 µl

 

  • Distribute 34 µl of the reaction mix with a micropipette and filtered sterile tips in each tube or well of a microplate for real time PCR.
  • Add 12 µl of sample of extracted nucleic acid or RNA positive control for the positive control or Elution buffer for the negative control.
  • Close immediately with an adhesive fillm to avoid all contamination
  • Centrifuge briefly to collect all the PCR reaction mix at the bottom of the tubes or plate.
  • Run the following Program on the qPCR instrument for the reverse transcription:

 

program Temperature duration Cycle (s)
Reverse transcription 42°C 30 min 1

 

Once the reverse transcription is done, the Taq N is added into each well.

Dilute the Taq N as follows by multiplying the number of N sample to test ( including positive, IPC, negative controls). On average, prepare enough reagents for N+3 reactions minimum.

Number of reactions (N) 1 N+3
Taq N 2 µl (N+3)x 2 µl
Elution buffer 8 µl (N+3)x 8 µl
Total volume of Reaction mix 10 µl (N+3)x 10 µl

 

Distribute 10 µl of the diluted Taq N with a micropipette and filtered sterile tips in each tube or well of a microplate for real time PCR.

  • Run the following Program on the qPCR instrument for the PCR amplification:
program Temperature duration Cycle (s) Fluorescence acquisition
DNA denaturation 95°C 5 min 1
Amplification 95°C 15 s 42
60°C 40 s yes
Cooling* 14.8°C 2 min 1

*temperature of the cooling step can vary according to the qPCR instrument. Please contact ADNUCLEIS for further information.

Note 1: set heating and cooling rate by default, up to 20°C/sec, or 100 %

Note 2: For Applied Biosystems, select “none” in “Passive reference”.

Note 3: n RotorGene, please calibrate the signal by clicking n “GAIN OPTIMIZATION”

IX.               Validation of the experiment

For the assay to be valid, the Ct values for the controls must be the following.

Outside of these values, the experiment is not valid.

Controls PRRS NA RNA control IPC Negative control
FAM <30 >35
VIC <37 >35

X.               Data analysis and interpretation

For clinical samples, the following results are possible:

FAM signal (target) VIC signal (IPC) Presence of the target Test validity/comment
<35 >37 positive
<35 <37 positive
Not Ct or >35 <37 negative valid
Not Ct or >35 Not Ct or >37 inhibited Possible inhibition of RT-PCR. Dilute ½ or 1/10 the extract, otherwise extract once again the sample and retest bt RT-PCR

XI.               Performances analysis

XI.1        Studies of repeatability and reproducibility

Studies of repeatability and reproducibility of the kit ADNUCLEIS PRRS NA virus have been achieved using a synthetic RNA by in vitro transcription on a plasmid containing a fragment of the PRRS NA virus.

The tables below indicate the coefficient of variation (CV) depending on the concentration of the RNA

 

Table of Repeatability (intra-experiment) and reproducibility (inter-experimental)

copies/PCR Répétabilité intra expériementale Reproductibilité inter experimentale
1.55E+08 1.55 2.43
1.55E+07 0.17 1.92
1.55E+06 0.35 0.75
1.55E+05 0.38 0.31
1.55E+04 0.50 2.52
1.55E+03 0.64 1.31
7.76E+02 1.97 2.28
1.55E+02 2.28 4.40
7.76E+01 0.09 0.82
 
Moyenne CV 0.88 1.86

 

XI.2        Performance analysis

  • Analytical sensitivity: 3.38×101 copies/PCR for the PRRS NA virus
  • Linearity for quantification: from 7.76×102 copies to 1.55×108 copies per PCR

*average Ct are calculated with the three replicates

kit Channel Efficacy coefficient of correlation R² Slope
SDRP-NA FAM 126.04 % 0.9941 -2.83

*average efficacy, R² and slope are calculated with the three replicates

 

 

XII.               Biobliography

  • Brar M. S., Shi M., Murtaugh M. P., Leung F. C. (2015). Evolutionary diversification of type 2 porcine reproductive and respiratory syndrome virus. J. Gen. Virol. 96, 1570–1580.
  • Dekkers J, Rowland RR, Lunney JK, Plastow G. Host genetics of response to porcine reproductive and respiratory syndrome in nursery pigs. Vet Microbiol. 2017 Mar 22
  • Gillespie, J., Opriessnig, T., Meng, X.J., Pelzer, K., Buechner-Maxwell, V., 2009. Porcine circovirus type 2 and porcine circovirus-associated disease. J. Vet. Int. Med. 23, 1151–1163.
  • Hansen, M.S., Pors, S.E., Jensen, H.E., Bille-Hansen, V., Bisgaard, M., Flachs, E.M., Nielsen, O.L., 2010. An investigation of the pathology and pathogens associated with porcine respiratory disease complex in Denmark. J. Comp. Pathol. 143, 120–131.
  • Holtkamp DJ, Kliebenstein JB, Neumann EJ, et al. Assessment of the economic impact of porcine reproductive and respiratory syndrome virus on United States pork producers. J Swine Health Prod. 2013;21(2):72-84.
  • Zimmerman JJ, Karriker L, Ramirez A, Schwartz K, Stevenson G. Diseases of Swine. Oxford: Wiley-Blackwell Publishing; 2012. pp. 461–462.

XIII.               Waste disposal

Dispose of waste according to DASRI regulations

Starting the QPCR program Taqman template

Rev 1.0 (29/06/2017)

How to run the QPCR Mx pro device:

  • Click the MxPro icon
  • A new window opens. Select “Quantitative PCR (multiple standards), then click OK. If the software is already opened, then click File -> New -> Quantitative PCR.
  • A new window opens -> do you wish to use Quantitative PCR plate setup adnucleis , click YES
  • The following windw opens -> do you wish to use Quantitative PCR Thermal Profile Setup, click YES.
  • Start the halogen lamp (for Mxpro QPCR device)

  • The lamp takes 20 min to warm up.
  • You can check the thermal profile by clicking on the tab Thermal Profile Setup. The thermal profile should be as follows: 95°C for 5:00 min, 42 cycles at 95°C during 0:10 sec / 60°C during 0:40 sec, then a step at 25°C for 2:00 min.
  • When the lamp is warmed up, click the RUN button.

  • On the RUN window, click “Start”  and name the file
  • Click “Save”.

The QPCR program starts (1h15 min)

  • When the run is done, save the data and export the data in excel file to be uploaded to ADN_SOFT :
  • Click on “Analysis” button

  • Click File->Export Instrument Data -> …To Excel ->Format 1…

  • Save the file and close it before to upload it into ADN_SOFT.

Results analysis using ADN_SOFT

Rev 1.0 (07/07/2017)

After opening ADN_SOFT

  • Click the “Import” button and follow the instructions.
  • Results will be displayed in two tabs. On on tab, results are displayed in a PCR plate view. On the other tab, results are displayed as a list.
  • Check in the “controls” tab that the positive, negative, IPC controls are ok.
  • Save the file as archive (Save As…)
  • Data can be exported in readable excel file (.xlsx)

NB : In case of a sample inhibition (“INHIB” ), follow the procedure below :
Dilute manually the RNA from the sample plate using the elution buffer. Add this diluted RNA in the next plate of extracted sample you want to test with the amplification mix. Please write this new sample in the plate layout of ADN_SOFT for the preparation of the nex PCR plate.

qPCR plate preparation – Robotic method

Rev 1.0 (29/06/2017)

Reagents and Materials included in ADNucleis kit :

  • The RT-PCR kits are distributed in bulks of 48 reactions under the form of a 2X concentrated premix.
  • Premix: The dilution of the 2x premix is managed by the robot using ELU buffer.  The 2X concentrated PCR premix contains all the ingredients necessary for the detection of the target (s) of the PRRS virus (primers, dNTPs…) and the IPC primer/probes, except enzymes.
  • Taq (Taq polymerase Nucleis used to amplify the DNA)
  • IPC / IAC _ARN  (RNA inhibition control)
  • RT (Reverse transcriptase Nucleis used to transcribe RNA into cDNA)
  • Elution buffer ELU
  • RNA positive control  (200µL/ tube of positive control is provided for each vial of 48 reactions)

Reagents and Materials not included in ADNucleis kit :

  • Recommended consumables
    • 96 well PCR Plate PCR (Be careful to use a plate suitable for both the Inheco device and your qPCR thermocycler)
    • QPCR Sealing film (e.g. ThermalSeal RT2RR)

  • Upon reception, ADNucleis amplification kits must be stored at -20°C until use.
  • DO NOT freeze back or reuse a reconstituted PCR 1x mix.
  • The reverse transcription step to generate cDNA from RNA is performed separately from the PCR. The RT step is carried out first by adding the RT enzyme in the premix manually. When the RT step is done, the Taq polymerase Nucleis is added t the mix by the robot in each well.
  • ADNucleis recommends to avoid freeze/thaw cycles (Not >3) of the amplification kits.
  • Vials of PCR premix  can be aliquoted to avoid excessive thawing and freezing when premix are thawed for the first time (8, 16 reactions). Aliquoted premix must be kept at -20°C.
  • If the component of the kits are not aliquoted, please write on the original  vials the number of thaws and volumes pipetted.

Example of annoted vials

Numbers indicate  the number of reactions  used  16, 12 and 4 puits; indicating 3 different thaws.

PROCEDURE

Generate a PCR layout and the worklist using ADN_SOFT

 

  • Choose the desired target (s) (SELECT_TARGET), and write an “x” in each well to be analyzed with the corresponding target.
    • For detection mode, the genomic quantification can be left blank. The bottom of the table shows the fluorescent channel used for each target.
  • Create the layout code for the robot by clicking “Create PCR plate(s)”
  • Fill the cell plate number (If there are several PCR plates to run all the samples extracted, the PCR plate can be generate successively by indicate the plate number). When 94 samples are tested with one target, only one PCR plate will be created (94 + 1 negative control and 1 positive control = 96 used wells). Fill the cell with “1”.
  • Generate the worklist by clicking “Generate Code”

Amplification mix reconstitution

  • Work with ice or cold bloc. Inheco blocks can be used; To use the inheco device manually: Click “Temp” if the temperature is already set to 4°C. Otherwise, click “set”, then “4” and “0”, then click on the left top button when the temperature is written “004.0°C”. Then click “Temp”. The button “Set” should indicate 4°C.
  • If extracted samples have been stored at 4°C or frozen before the purification step, the deepwell containing samples should warm on the thermoshaker “Inheco DW 96 Ext” of the robot set to 4.0°C and thaw for 10 min at this temperature.
  • Thaw HQS_xxx_PRE-mix premix vials, IPC vials and the RT Nucleis vials using the 24 well Inheco block of the robot. Put the vials in each slot according to the map given by the ADN_soft. Put an empty vials to reconstitute the amplification mix.
  • The mix reconstitution is performed according to the table below
 Component Volume per well (µl) Volume per Y well (µl)
HQS_xxx_pre-mix (with IPC) 18 18 x Y
RT Nucleis 1 1x Y
IPC 3 3 x Y
reconstituted Mix(µl) 22 22 x Y

NB: ADN_SOFT will show the different volumes for the mix reconstitution. The software  takes into account the number of reactions and loss of reagent due to pipetting. Volumes of reagents provided by ADNUCLEIS in each kits take into account this loss of reagents due to pipetting. The 48 reactions kits contain enough volume for 58 reactions).

BE CAREFUL :

  1. As well as for PVG, NO VOLUTE should remain after homogenization. Pipet 10 times in order to homogenize correctly Premix with RT and then with IPC.
  2. No bubbles should be seen inside tubes (reconstituted mastermix , positive rna, Taq)

Starting the robot and QPCR

  • Put a  1.5 ml vial into the slot indicated by ADN_SOFT, add the volume of Taq given by ADN_Soft.
  • Add 10 ml of ELU buffer and 20 ml of D1 buffer into the indicated container.
  • Check if the liquid system container contains enough volume of 2% D3. Fill it if necessary according to the volume indicated by ADN_SOFT. Be careful when filling the container. Add the water first, then the adequate volume of concentrated D3 to avoid foaming.
  • Empty the waste container
  • Verify the reconstituted mix, Taq, RNA positive control vials are correctly placed according to the 24 well Inheco device map on ADN_SOFT.
  • Put a new PCR plate on the PCR Inheco device.
  • Click “flush” in Gemini

  • Then, click “initialize”

  • To run the program click file and choose the program “ADNucleis_qPCR_plate_prep”, then click “Play”
  • Follow the program
  • At the end the run (around 30 min + 1 min per sample), seal the PCR plate and put it immediately in the QPCR apparatus and launch the QPCR program. If you do not run the QPCR immediatly, then freeze immediatly the PCR plate at -20°C until use.

RNA Extraction – Purification – Robotic method (96R Ext_ARN PVG100+ Purification BM)

Rev 1.0 (29/06/2017)

Reagents and Materials included in the ADNucleis kit:

  • ADNUCLEIS Viral RNA Extraction-Purification kit (Extraction kits and Purification kits are sold separately)
    • RNA extraction kit: PVG working solution must be prepared before starting the extraction step)
      • PVG (Please reconstitute the solution in this bottle)
      • PE
      • TME
      • RNA carrier (Keep at 4°C)

(Please use immediately after preparation).

  • Nucleic acid Purification kit

    • Magnetic beads suspension (µMB)
    • Precipitation buffer SP
    • Wash buffer 1
    • Wash buffer 2
    • Wash buffer 3
    • Elution buffer (ELU)
    • Decontamination buffer for the robot needles: D1, D2 et D3

Reagents and Materials not provided in the kit

  • Consumables
    • Deep-well 96 wells plate (for the robotic method,  please use the following plate: Riplate plus PP 1mL ref. n°43001-0016, Ritter brand).
    • Aluminium foil film for 96 wells plate.
    • 96 wells PCR plate
    • QPCR film for 96 wells PCR plate

The material above  is not included in the kit, but can be purchased at ADNucleis. Please contact ADNucleis to check the compatibility of your own consumables.

  • Routine laboratory reagents and materials (to be used according to the Good Laboratory Practices): tips, micropipettes (1mL, 200µL, 20 µl, 10 µl), non-powdered gloves …
  • Ultrapure water (sterile, distilled and nuclease free).
  • RNA Standard.

RNA extraction is achieved using enzymatic activities: An enzymatic and thermal lysis releases the RNA from all microorganisms and cells present in the sample. .

Preparation of the PVG working solution :

  • Resuspend PE with the appropriate volume of TME (ME) when first opening the PE vial. Homogenize
Reagents  TME volume
PE 25 mg 440 µl
PE 100 mg 1760 µl

Store at -20°C until use.

  • Prepare enough master working solution of PVG + ME (100µL of PVG for 4µL of ME) extemporaneously for X number of samples to be extracted +3 (e.g. for 20 samples, prepare a volume of master mix for the equivalent of 23 samples).
  • BE CAREFUL TO HOMOGENIZE CORRECTLY THE PVG in order to have NO volute in the end (10 times).
  • RNA carrier should be added in the mix PVG + ME at this step if extracting RNA. Add 1 µl of RNA carrier per sample (e.g. 23 µl)
Reagents 1 reaction 24 reactions 48 reactions 72 reactions 96 reactions
PVG 100 µl 2700 µl 5100 µl 7500 µl 9900 µl
ME 4 µl 108 µl 204 µl 300 µl 396 µl
RNA carrier 1µl 27 µl 51 µl 73 µl 99 µl

Volumes in the table above for 24, 48, 72, 96 reactions take into account the loss of volume due to pipetting. ADN_SOFT takes into account the loss of volume due to pipetting automatically.

  • Distribute 100µl of Extraction buffer PVG + ME (+RNA carrier if added) in each well of the deep-well plate.
  • For PIPETTOR ROBOT automated procedure, this distribution is done column by column beginning with the first column (A1, B1…H1, A2, B2…H2 etc.)

    

It is possible to have the first sample placed in any well (Offset option). However, the filling of the following samples (2,3,4,…) must follow the same rule as above: samples are distributed column by column from top to bottom and no empty well is allowed between the first and last sample. The number of empty wells before the first sample is given in the “plate Layout” sheet.

  • Homogenize samples carefully, then pipette 100µl and add to a well of the deep well plate.
  • Vortex or homogenize by pipetting the sample with the PVG.
  • Put the deepwell plate with samples on the heating block (Thermoshaker)
  • Start ADN_SOFT.exe (“START A NEW RUN”), and click immediately on “Save As..” and save the archive file in the default folder.
  • fill the plate layout with sample names (each name has to be different), then click on “Lock Layout”.
  • Specify the volume of elution buffer used for the RNA elution step.
  • The table below indicates reagent volumes to be filled in the different containers of the robot.
  • The program used for the Extraction-Purification method is “µMB Extract-Purif” :

  • Check that the volume in the Liquid System jar is sufficient (2% D3 in demineralized water). Empty the “Waste” jar.
  • Start Gemini software (Software controlling the Tecan Robot), then open the extraction purification program : ADNucleis_Extract_Purif.gem
  • Fill the different containers with reagents according to the positions showed on the Layout in Genimi software (Required volumes are given by ADN_SOFT).
  • Put an empty deepwell plate in “DW-Empty” position, This plate will be used to collect eluted RNA.
  • Start the Inheco Multitec heating block. The Gemini program controls the temperatures of the different blocks.
  • Click “flush” on Gemini software

  • Initialize the robot

  • Click “Play” in Gemini software to launch the extraction-purification program. A prompt window will ask the number of sample, fill the number of sample.

NB : the robot will start by the thermic lysis step. This steps takes about 30 min. The robot does not show any visual activity.

  • At the end of the program (30 min + 1min per sample), the RNA is eluted and the plate is kept at 4°C.
  • Go back to ADN_SOFT to start the setup of the PCR plate

 

Sample preparation – Semen

Rev 1.0 (29/06/2017)

Boar semen samples should be kept between 1 and 4 °C or on ice until use.

Raw semens are first diluted 1/10 in a buffer (Elixir buffer -genePro).

100 µl of the diluted semen are used for the nucleic acid extraction.

Troubleshooting – Robot

In case of error, initialization error, please check that there was no collision done by a bad positioning of tube, rack, plates… As the robot initialize its position on each start, it is possible to clear problem by switching off the robot (wait 3 sec) and re switching on. Restart Gemini and initialization.


« Device is not implemented », happening during initialization

Cause :

Can be caused by a wrong initial position which causes an mechanical problem.

 

Corrective Task :

Turn off Pipetting Robot, move the Robotic Arm on any another position of X, Y and rotation, then re launch Pipetting Robot and Gemini software.


Diluter error “tip is broken”

Cause : This problem can occur when a wrong dispense operation has been done. The robot or the operator “Desactivated “ the tip.

Corrective Task : 

To repair this, Setup -> Configuration -> LiHa -> correct the check box where the tip is marked as broken.


Diluter error “Plunger overload”

The robot stops until operator click on “initialize”. Once re initialized (click on Initialize), the robot can continue the run. If the robot doesn’t succeed in initialization procedure, switch off the robot, check if needle is not clogged, move diluter (the screw under syringe) up and down and retest.

Cause : 

This error can mean that needle is blocked or diluter is damaged.

 

Corrective Task :

If the problem occurs more than 1 time each 5 runs on the same channel (which would be not plugged), diluter has to be changed. Procedure is as follows :

0- Switch off the robot, set the robotics arms in the middle in order to be able to lift the hood.

1- Unscrew bottom syringe screw

2- Put down the arm

3- Unscrew the syringe, set it on the other new diluter

4- Unscrew the 2 top tubes – BE CAREFULL OF WATER WHICH COULD DROP DOWN FROM THE 2 TUBES TO ELECTRONICS COMPONENTS OF THE ROBOT/DILUTER (you can put tubes on a wipe to absorb water.)

5- Check below the diluter if any screw retains diluter, and if there is one, unscrew it too.

6- Remove the old diluter

7- Check the address behind it, and report the same on the new one (1st diluter is “0”, 2nd is “1”, etc.)

8- Set the new one, – BE CAREFULL OF WATER WHICH COULD DROP DOWN FROM THE 2 TUBES TO ELECTRONICS COMPONENTS OF THE ROBOT/DILUTER

9- Screw firmly tubes, then syringe, then arm syringe.

10- Start robot, start Gemini and initialize. If error occurs, recheck address increment is correctly set.

 


Detection error “no liquid”

 

Cause :  

can be caused by :

–  Not enough liquid in the tube/troughs

–  Deionized liquid in the tube/trough so the robot cannot detect it.

–  ADN_SOFT underestimated the needed volume (in this case, please contact support@adnucleis.com ),

–  Evaporation of the liquid because too much time occurred between filling and program run.

 

Corrective Task

Please refill the liquid, and click on “Retry Detection”.

NB : If the volume inside looks “OK” for the estimated needed aspiration but the volume is so low that the robot can’t detect it, you can click on “Move tips to Zmax”, the robot will aspirate to the bottom of the tube / trough.

Reagents and plate setting

 

 

Slot :

 

 

Phasis :

ORIGIN ELU D1 E D2 SP W1 W2 W3 µMB DeepWell including samples on Thermoshake  

 

 

Empty 96-wells Deepwell

(And binding columns plate depending on installed technology)

Mix Tubes on Inheco with tubes adapter

 

qPCR plate on Inheco with pcr adapter

 

Extraction

ADNucleis_EXTRACT.gem or ADNucleis_EXTRACT_DIL*

Clean Waters

 

90 mL* 30 mL

 

15 mL  

X

Extraction Purification

ADNucleis_EXTRACT_PURIF.gem

All other matrices 15 mL 30 mL 15 mL Cf ADN_SOFT Cf ADN_SOFT Cf ADN_SOFT Cf ADN_SOFT Cf ADN_SOFT  

X

(The plate contains samples inside 100µL F and 100µL PVG buffers)

X
Extraction Purification COLUMNS

ADNucleis_COL_EXTRACT_PURIF.gem

All other matrices 15 mL 30 mL Cf ADN_SOFT Cf ADN_SOFT Cf ADN_SOFT Cf ADN_SOFT  

X

(The plate contains samples inside 100µL F and 100µL PVG buffers)

Seal partially the silica columns plate where unused wells :

 

 

 

X

PCR plate preparation

ADNucleis_qPCR_plate_prep.gem

Extract w/wo Purif 15 mL 30 mL  

X

(The plate contains eluted DNA Deepwell)

 

X

(cf program for ref and positions)

 

X