Πρωτόκολλα

1 . Απομόνωση RNA από ιστό

1. Place the tissue in 1 ml Qiazol Lysis Reagent. Full speed for 20-40 sec. Let the homogenate at room temperature for 2-3 min.
2. Place the tube on the benchtop at room temperature for 5min.
3. Add 200?l chloroform. Shake it for 15sec.
4. Place the tube at R.T for 2-3min.
5. Centrifuge at 12000g for 15min at 4oC .
6. Transfer the supernatant in a new tube.
7. Add equal volume of chloroform.
8. Centrifuge at 12000g for 5min.
9. Transfer the aqueous phase to a new tube. Add 1volume (600?l) 70% ethanol. Vortex. DO NOT CENTRIFUGE.
10. Transfer 700 ?l of the sample to an RNeasy column placed in a 2ml collection tube. Centrifuge 15 s >10000rpm. Discard the flow through.
11. Dissolve the DNase I (stock solution) in 550 ?l of RNase-free water. Mix well, DO NOT VORTEX.
12. Pipet 350 ?l BUFFER RW1 into the RNeasy mini column. Centrifuge 15sec >10.000rpm. Discard the flow-through.
13. Αdd 10 ?l DNase I to 70 ?l BUFFER RDD.
14. Pipet the DNase I incubation mix (80 ?l) directly onto the RNeasy silica-gel membrane. Place on the benchtop for 15min.
15. Pipet 350 ?l BUFFER RW1 into the RNeasy mini column. Centrifuge 15sec >8.000 g. Discard the flow-through.
16. Transfer the RNeasy column into a new 2ml collection tube. Pipet 500 ?l BUFFER RPE onto the RNeasy column.
Centrifuge 15sec >10.000rpm. Discard the flow-through.
17. Add 500 ?l BUFFER RPE to the RNeasy column. Centrifuge 2min >10.000 rpm to dry the RNeasy silica-gel membrane.
18. Place the column in a new 2ml collection tube. Centrifuge in a microcentrifuge 1min at full speed.
19. Transfer the column to a new 1.5ml collection tube. Pipet 40 ?l RNase-free H2O onto the RNeasy silica gel membrane. Centrifuge 1min >10000rpm.
20. Repeat step 19.

2. Πολλαπλασιασμός του mRNA και σήμανση

Reverse Transcription - First strand

Materials
OligoT primer, 5' MC-T7-(dT)24V 3', 65mer (100 ng/ μl)
5X First Strand Buffer (Superscipt)
0.1 M DTT
dNTPs (10 mM each)
Superscript III 200u/μl

• Place on 0.2 ml tube 4 μ gr total RNA in final volume 11.5 μl max
• Add 1.5 μl OligoT primer, 5' MC-T7-(dT)24V 3' , 65mer (100 ng/ μl)
• Adjust volume to 13 μl with DEPC water ( if necessary)
• Incubate at 70ο C for 10 min ( denaturizing )
• Place on ice for 1-2 min and do quick spin if there are vapors on the walls of the tube .
• Prepare mix (without the enzyme) and prewarm it at 50ο C for 5 min
• Add 7 μ l mix: 4 μl 5 x First strand buffer
1 μl 0.1 M DTT
1 μl dNTPs (10 mM each)
• Add the mix to RNA
• Add 1 μl Reverse Transcriptase (Superscript III 200u/ μl)
• Incubate: 20 min at 44ο C
1 h and 45 min at 50ο C
15 min at 75ο C ( superscript III inactivation)
• Proceed directly to the second strand cDNA synthesis (Step 4)

Second strand cDNA synthesis

Reaction (100 μl final volume )
20 μl reverse transcription reaction (If less than 20, add DEPC-water)

Reaction mix (prepared on ice )
55 μl DEPC water ( on ice )
20 μl 5 X second strand buffer
1.5 μl dNTPs (10 mM each)
2 μl DNA pol I (10 u / μl)
1 μl RNase H (2 u/ μl)
0.5 μl E.coli DNA ligase (10 u/ μl)

• Incubation at 16ο C for 2 hours
• Addition of 1 . 5 μl Τ 4 polymerase (3 u / μl )
• Incubation at 16ο C for 15 min
• Storage at -20ο C or cDNA purification

The addition of DNA ligase and Τ 4 polymerase is not necessary , as their absence does not affect either the final quantity, or the quality of amplified RNA that is produced . ( see appendix ).

cDNA purification

• Add DEPC water till volume 200 μl
• Add 200 μl PhOH/CH 3 Cl/Isoamyl alcohol (25:24:1) Mix , centrifuge at 12.000 rpm for 5 min.
Transfer aquatic phase to a clean tube
• Add 200 μl CH 3 Cl / Isoamyl alcohol (49:1) Mix , centrifuge at 12.000 rpm for 5 min. Transfer aquatic phase to a clean tube
• Centrifuge again at 12.000 rpm for 5 min and remove organic phase ( if there is some )
• Add : 100 μl 7,5 Μ ammonium acetate
1 μl glycogen (20 μg /μl )
400 μl isopropanol
• Mix well and incubate for 10 min at room temperature
• Centrifuge at 12.000 rpm for 20 min
• Discard supernatant and wash the pellet with 75% EtOH
• Proceed or store at -20ο C
• Centrifuge at 12.000 rpm for 5 min and remove EtOH
• Dry the pellet on air
• Proceed to in vitro transcription

In vitro transcription

NOT in 1.5ml tubes (significant evaporation for >10h)
Preferably in 0.2ml tubes

Reaction (20 μl final volume)
7,25 μ l H 2 O (sterile, RNase-free from the kit!)
2 μl 10 x T7 buffer
1,5 μl ATP (100 mM)
1,5 μl GTP (100 mM)
1,5 μl CTP (100 mM)
0,75 μl UTP (100 mM)
1,5 μl aaUTP (50 mM)
2 μl 0.1 M DTT
2 μl T7 polymerase (NOT IN MIX)

• 10x T7 buffer is prewarmed at 37ο C/10 min to avoid the formation of any precipitants
• Reaction mix is prepared at room temperature

Resuspend pellet from cDNA purification in 18 μl reaction mix
Add the enzyme (2 μl)
Incubation at 42ο C (or 37ο C) for 7 hours

Electrophoresis in agarose gel (1-2 μl from the reaction)
Purification of aaRNA with RNeasy mini kit
Quantification with Nanodrop .

The quantity of amplified RNA that is produced with this protocol is usually about 15 μgr ( initial total RNA is included ).

Cleanup of IVT RNA with RNeasy columns

Prior procedures

Before using buffer RPE for the first time, add 4 volumes (44ml) of absolute ethanol (under hood) and mark it.

All steps, including centrifugations, should be performed at RT (room temperature).

1. Take the appropriate volume of buffer RLT ( 0.35 ml for each IVT). If a precipitate is formed, redissolve by warming (37o C) and place at RT. Contains guanidine salt-irritant, wear gloves.
Add before use β -mercaptoethanol (10 μ l per ml of RLT buffer, stored in the fridge . Careful toxic, hood and gloves). Stable for 1 month after that.
2. Adjust sample to 100 μ l with DEPC-treated water. Add 350 μ l of buffer RLT (with β - ΜΕ ) and mix thoroughly.
3. Prepare the RNeasy column placed in a 2ml collection tube (kit). Mark the top of the column, but not the side, as it may be washed by ethanol. Then add 250 μl absolute ethanol to the sample and mix well by pipetting. Do not centrifuge, as the nucleic acids are in a semi-precipitated state.
4. Apply the sample (700 μl), included any formed precipitate, to an RNeasy mini column. Close the tube gently. Centrifuge for 15 sec at full speed. Pass the flow-through through the column one more time and discard the flow-through and the collection tube.
5. Transfer the RNeasy column into a new 2 ml clooection tube (kit). Pipet 500 μ l buffer RPE (with ethanol) onto the RNeasy column. Close the tube and centrifuge for 15 sec full speed. Discard the flow-through, but reuse the collection tube.
6. Add another 500 μl buffer RPE (with ethanol) onto the RNeasy column. Close the tube and centrifuge for 2min full speed. Discard flow-through and collection tube.
7. Recentrifuge in a 1.5ml tube (not in kit) for 1 min. Discard flow-through and collection tube. Put column in an RNase-free 1.5ml tube (kit). Mark the top of the tube.
8. Apply 40 μl RNASE-FREE water (kit) in the column and incubate at RT for 2mins. Elute by spinning at full speed for 1 min. Repeat elution with another 40 μ l RNASE-FREE water. Put tube on ice . Don't discard the column till after the quantitation.

Quantity and Quality

Because RNA can form extensive secondary structure, the most accurate way to determine the size distribution is via denaturing gel electrophoresis. This most commonly includes either agarose gel electrophoresis of glyoxylated RNA or formaldehyde denaturing agarose gel eletrophoresis of formamide denatured RNA. The primary drawback to gel electrophoresis is that it requires microgram amounts of RNA.
A non-denaturing agarose gel can provide a good estimate of the size distribution of RNA if the RNAs and RNA molecular weight ladder are both denatured in a formamide loading buffer.The amplified aRNA should range from 250-2500 nt with the median of the distribution centered around 750 nt.

Materials

10X TAE / 1X TAE
Denaturing Gel Loading Buffer
(95% formamide, 18mM EDTA, 0.025%SDS, Xylene Cyanol and Bromophenol Blue)-950 μ l formamide, 36 μ l 0.5M EDTA, 2.5 μ l 10%SDS, 11.5 μ l water
High Quality agarose
DNA Ladder (in DNA loading buffer)
ethidium bromide stain (1mgr/ml)

Procedure (adapted from Enzo):

1. Determine the RNA concentration by the Nanodrop (2 μl in 2 μ l TE pH8).
2. For 100 ml gel, add and melt 1-1.2 g of agarose in 100 ml of 1X TAE and cool to ~60°C. Add ethidium bromide 30 μl (from 1mgr/ml stock).
3. Pour the gel and allow it to set. The wells should be large enough to accommodate 20 ?L.
4. While the gel is setting, transfer 1 μ g of each RNA sample to a microcentrifuge tube on ice. Include an DNA ladder sample (1 μ gr ). Add nuclease-free water to bring the volume of each RNA sample to 6 ?L. Add 12 ?L of Denaturing Loading Buffer, mix, centrifuge briefly and heat at 65°C for 5 minutes.
5. While the RNA is being denatured, remove the comb and place the gel in the gel tank. Add enough 1X TAE to cover the gel.
6. After denaturation, place the RNA samples on ice and then briefly centrifuge.
7. Load the samples and run the gel first at 70V and then at 110V, until the bromophenol blue dye has migrated about two-thirds of the length of the gel.
8. Carefully remove the gel and place in clean staining tray

While the gel contains no denaturant, we find that this system behaves more like a formaldehyde gel than a native gel. The size range of the RNA can be estimated by the DNA ladder.

Labeling

( We do the whole process in dark , at room temperature)

Reaction (20 μl final volume)
8 μl aaRNA (5-20 μ gr ) if the volume is less than 8 μl we adjust it with DEPC water
2 μl 0.5 M sodium bicarbonate pH 9
10 μl DMSO

• Warm the mix at 500 C for 5 min
• Transfer the above 20 μl of our sample to the dry dye pellet and re-suspend via pipeting . DO NOT spin .
• Incubate for 2-2,5 hours in dark , at 500 C
• Increase volume to 25 μl
• Clean up labeled RNA with G 50 column . Repeat the cleaning.
• Measure absorbance and calculate dye incorporation.

Dye incorporation is usually 1 molecule dye per 40 bases (40 base / dye ). For hybridization , 3 μgr of labeled RNA with a ratio base / dye 40 are enough to give a satisfactory signal.

3. Πρωτόκολλο υβριδοποίησης στον σταθμό TECAN

Read the manual of the Tecan HS 4800. Take special care in the maintenance chapter.

Materials

Printed slide
Labelled samples (R, G)
Blocker (fragmented salmon sperm DNA 10 μgr/ μ l)
1.3* Hyb buffer (for 100 ml: 25ml 20*SSC (RNA grade), 1ml 10%SDS (RNA grade), 50ml formamide. a Filter sterilize and aliquot).

Tecan preparation

Put the slide in an empty chamber. Be alerted for dust, glass fibers and rubber wear. Close the frame carefully.
Check that there is enough liquid in all 4 buffers.

Buffer 1: 5*SSC, 0.1%SDS
Buffer 2: 2*SSC, 0.1%SDS
Buffer 3: 0.1*SSC, 0.1%SDS
Buffer 4: 0.1*SSC

The Buffer 1 is precipitated in RT. If so, warm it in the microwave.
Connect the hyb station with the PC attached to it. Turn on the reagent heating. Open the appropriate "Signed" hyb protocol.
Put the tubings in the correctly numbered bottles
Prime the appropriate modules, using channel 1.

Procedure

Protect labeled samples from light

Turn on the nitrogen valves. Check the pressure to be 2.7 atm.
Start the signed protocol.
Warm 60 o C, degas and warm again the prehyb buffer (5*SSC, 0.1% SDS, 1%BSA).
When prompted, inject 100 μ l to the corresponding slides (Set the pipette to 106 μ l, pipet till the first level).
Speed vac the differently labeled samples separately, using an aluminum foil cover to 18 μ l.
Combine them. If needed, fragment them with 2 μ l of Ambion fragmentation buffer for 15min at 70 o C and stop it with 2 μ l of Ambion stop buffer.
Add 1.5 μl salmon sperm DNA per hyb.
Adjust volume to 27 μ l with RNA-grade water. Add 85.5μl Hyb Buffer.
15 min before hybridization, prewarm in 60 o C degas and warm again.
10. Inject the probe.
11. After the hyb is over, turn on laser 1 and 3 in the scanner (for R and G channels).
12.Open the frames SLOWLY.
13. Put the slides in an appropriate dark container.
14. Turn off reagent heating.
15. Put dummy slides in their place. Put all tubings in the Blue bottles filled with water and cover the top of the Red bottles with aluminum foil.
16. Rinse the used modules, enabling final system drying.
17. When the sound starts, put the tubings in an empty container and press enter.
18. When rinse is complete, turn off the nitrogen valves.
19. Open and detach the frames. Clean them, as described in the manual and dry them with pressurized air.

4. Σάρωση της μικροσυστοιχίας και ανάλυση εικόνας

Scanning

Read the Scanarray manual .Scan only slides with standard dimensions (1"x3")

Turn on the scanner
Wait till it passes the startup checks and the standby indicator turns green.
Connect to the scanner with the Scanarray program from the PC.
Turn on the lasers (15min to warm).
Eject the slide holder.
Put the slide carefully in. If you don't feel resistance, close the slide holder and eject it again.
Default scanning protocols are stored under the Protocols tab. If you want to create a new one, follow the manual instructions and the stored examples, also include scanning in Laser Power 90% and PMT 75%, 65% and 55%
Make a quick scan at 50 or 30 μ m resolution. Check the boundaries and use the Line Scan tool in a bright area to focus the scanner.
Under Focus/ Line scan mode, make a Start Scan to check that the area is bright and then Autofocus. Check that the focus distance is consistent.
10.Change the scanning resolution to 5 or 10 μ m, according to your slide.
11. Start scan
12. After scanning, check your images and save them in a unique folder named after the user and the date of scanning.
13. Turn off lasers , exit program and then shut down the scanner.

Useful files - File management

Image files

Must be saved in a unique folder named after the user and the date of scanning.
Temporary storage in shared folder Thanos in the Desktop. Scans older than a month may be deleted without prior information.
Move them to your Dataset folder.
Back up your images in DVDs.
To save space, scans and analysis older than a year can be backed up in DVDs

Array grids and templates

Ask the person in charge to provide you with the appropriate files
If there are not available (eg not standard chip), make your own, according to the manual instructions and the already available files as examples

Image analysis (with Imagene)

Read the Scanarray manual

Open the Imagene program *** in the Workstation PC.
Settings are project/slide specific. If you are the first to check a new type of array, use the settings stored as default the given time, check the results (requires experience and careful reading of the manual) and modify them accordingly.
Open the image files (all 6 of them) from your Dataset folder.
Adjust brightness and Contrast using the slider, so that you maximize the information content (able to see the weak spots without seeing the background).
Focus in a single bright spot. Use the alignment tool and check the result.
Load the template or the grid.
Move it over their correct positions, first globally with the Metagrid tool , then per block with the subgrid tool , next to it.
Change in the grid properties the min and max diameter (measured in pixels) close to the ones in your array.
Address the spots using the Auto-Adjust spots button
Manually mark the bad spots , especially those that are in regions with high background. This gives them flag 1, while flag 0 is for the good ones. The most important step (if not sure, ask for help).
After you check the result, press Quantify.
Rank the spots according to the flags. Check all the 2s and 3s for bad spots and mark them.
Store the results in the same folder as your images, under a 'DATE'raw folder (eg 310105raw for 31 January 2005).

5 . Πρωτόκολλο επίστρωσης πλακιδίων με APTS

( for 25 slides )

A SLIDE CLEANING

Always use no powder gloves
Label slides on back side before cleaning

1. Fill the slide racks with slides. Look at each slide and discard those that are visibly stained with streaks of dirt.
2. Prepare wash solution for the slides. Use 250 ml wash solution per slide jar. The wash solution is made of 50g NaOH pellets dissolved in 200 ml dd-Water and 300 ml EtOH 95% per jar.(Final vol 500ml , 250ml/ jar) This solution should be made fresh.( add EtOH slowly ,exothermic reaction)
3. Submerge the holder into the NaOH solution and shake gently (60rpm) for at least 2 hours (over night incubation is fine) . This solution should be made fresh every time as NaOH may oxidize. Use gloves while handling this solution.
4. From now on, keep slides submerged to avoid dust.
5. Rinse off the NaOH cleaning solution from the slides using distilled water. Place the slide holders in a big beaker and rinse them thoroughly under distilled water. Wash each slide holder for at least 3 minutes. After rinsing, keep them submerged in water.

B COATING SLIDES with APTS

1. Prepare the coating solution (250ml): 1% APTS in 95% EtOH (keep slides into ddH 2 O for avoiding the dust, meanwhile). Use only plastic ware
2. Transfer slides to coating solution.
3. Shake O/N at 60rpm.
4. Transfer to fresh chambers in ddH 2 O. Wash in H 2 O extensively. (a 4Lt Beaker is fine).
5. Spin dry slides @ 1500rpm for 5 min.
6. Wrap slide box in foil and put in a 80 o C oven for >2hrs (or even O/N)
7. Store slides in dessicator in closed slide box and label.

AFTER PRINTING

Put slides in humid chamber for 30-45 min (or rehydrate-snap dry), bake 4hrs @ 80 o C (or O/N), and UV crosslink them at 600 mJ .

Material

APTS : aminopropyltriEthoxysilane Fluka Cat No 09324
Slides : Gold seal slides Cat No 3010

6. Πρωτόκολλο ενεργοποίησης πλακιδίων με PDITC

PDITC ACTIVATED slides

Always use no powder gloves

Preparation of activated slides

1. Use (fresh) APTS coated slides
2. Submerge slides in activation solution .(0.2% w/v PDITC in 10% pyridine 90% DMF)
3. Incubate O/N shaking @RT.
4. Washes (10mins each): 2x DMF / 1x EtOH
5. Dry by spin or under a nitrogen stream.
6. Store in dessicator.

After the printing

1. Incubate O/N in a chamber under saturated humidity. (humid chamber=a closed box with 1cm saturated NaCl sln in bottom ,RH=75%) (spots up)
2. Bake slides a few minutes @ 80-100C to dry (spots up)
3. UV crosslink slides at 600mJ (spots up)
4. Store in dessicator. (Label box and fill in Logbook)

Activation solution (200ml per box)

• 0.2% (10mM) 1,4-phenylenediisothiocyanate (PDITC) 0.4g
• 10% v/v Pyridine 20ml
• 90% DMF 180ml

7 . Πρωτόκολλο εκτύπωσης πλακιδίων

Day before real print

1.Quality control of slides and printing procedure

2.Resuspend half of the plates of the real print run

CHECK QUALITY OF PDITC SLIDES

Use 2 PDITC activated slides from the batch you will use in the actual print.
Check for scratches dust and examine even coating by scanning at Cy3 Cy5 channels
Save their images in appropriate folder and fill in the excel PrintLog book

PREPARE PRINT PLATE

Thaw " QC-control " Genetix microtiter plate at room temperature for 15min.
Place plate in centrifuge and spin it down (1000-1500 rpm a few min) to collect samples on bottom
Carefully peel off the metallic adhesive covering
Using the Biomek, program " Rssp 1 print plate " , Resuspend oligos in Ultra Pure water to 10 uM (It's already resuspended in 1X spot buffer and dried ) Cover with costar sealing tape and shake it on platform shaker for >1hr to resuspend
Spin it down again to collect samples before print.

PRINTING

Check pins , printhead ,fluid level in wash bottle and humidifier bottle, and printing parameters are correct . If pins or printhead need cleaning clean them .
For preblot use 1 APTS or PDITC slide (not plain glass slide) and 2 PDITC slides for printing
Put slides-plates into spotter with correct orientation.
Run the printing protocol " QC before HOL print " using plate above

POST PROCCESSING

After print run, remove slides from spotter and place them on the metal support
Place slides + support in sealed humid chamber box. Place in the container a trough of water supersaturated with sodium chloride. The sludge of undissolved NaCl should be about 1 cm deep. (RH=75%)
Leave slides in chamber overnight.
Bake for a while to remove moisture (5min at 100C) then UV crosslink at 600 mJ
Store slides in dessicator .
Fill in the excel PrintLog book.

Allow QC-control plate to dry through passive evaporation in a protected environment or by speedvac then cover with aluminum seal and store at -20 .
For each subsequent preparation of this plate for a print run, add water to the wells to 10uM. The amount of water should be decreased by 0.25 ?L per print run, as this is the amount drawn up by the pin capillary during each dip
Evaluate spotting by red reflect scanning and hybridization
Save their images in appropriate folder and fill in the excel PrintLog book
Start resuspending half of the plates that you are going to print next day the same way you resuspend the QC control plate above but leave them shaking O/N sealed with aluminum tape

Day of real print

During the printing of the first 12 plates (that you have resuspend the previous day) you can continue resuspending with Biomek the remaining plates that you are going to print.(see prepare print plates above)

Check again wash-humidity fluid level, be sure you are using the correct protocol
Wash pins for 5 times.
Spin down 2 first plates that you were resuspending O/N (1500 rpm 10 min) remove cover and put in appropriate position inside spotter ( care A1 position)
Put 20 PDITC activated slides (care label - orientation ) and 4 preblot slides (APTS or PDITC)
Run the appropriate printing protocol e.g . HOLA
it takes ~1hr to print 2 plates so before change plates spin down the next plates
After a plate has been printed, remove it from the arrayer, allow plate to dry through passive evaporation in a protected environment (usually 1-2 days), then seal with aluminum sealing tape (Costar 6570) and store at - 20 .
Fill in the excel PrintLog book about plate usage and rssp volumes

HOL Printing Protocol

HOL is contained in 92 384 Genetix plates and is printed in 4 parts/slides named HOLA HOLB HOLC HOLD. Each part has 23 HOL plates +3 X SpikeIn Plate (SI) = 26 plates total in the following order : SI-11HOL-SI-11HOL-SI-1HOL.

The printing protocol is created with the following settings:

Plate set= HOLA (or HOLB or HOLC or HOLD ) No of plates per plate change=2 16 pins spotting at 180um spacing Array layout : custom, from left 3500um from top 8000um block=4500X4500 8 preprint spots at 350um Pin motion : Printing approach velocity =10mm/sec

// departure // =20mm/sec
// overtravel =370um
// dwell time =10mSec
Sample load overtrave =100um
// // dwell time =300um
Substrate thickness = 1mm
Max No of prints/load =120
Wash and dry : 3 sec wash X2 times, 3sec dry
Relative humidity =55% temperature 22C

Post Processing

1. After print run, remove slides from spotter and place them on the metal support
2. Place slides + support in sealed humid chamber box. Place in the container a trough of water supersaturated with sodium chloride. The sludge of undissolved NaCl should be about 1 cm deep.
3. Leave slides in humid chamber overnight.
4. Bake for a while to remove moisture (5min at 100C) then UV crosslink at 600 mJ
5. Store printed slides in dessicator (label slide box with print batch and print date).
6. Fill the Print LogBook.

8. Πρωτόκολλο ανίχνευσης 17 μεταλλαγών του γονιδίου της β-σφαιρίνης

Reagents required

NOTE: It is recommended that the following reagents (Manufacturer and Catalog Numbers when listed) be used.

β -globin PCR Primer Mix (*1)
β -thal 17 ASPE Primer Mix (*2)
β -thal 17 FlexMap Beads (31 populations)
2X Tm Hybridization Buffer (0.4M NaCl, 0.2M Tris pH8.0, 0.16%TritonX-100)
(TritonX-100, SIGMA T-9284)
HotStart Taq Master Mix, QIAGEN Cat. No.203445
dNTPs 100mM each
dCTP-biotin 0.4mM, Invitrogen Cat.No.19518-018
Platinum Tsp DNA Polymerase (5 U/?L) Invitrogen Cat. No.11448-024
(including 10X PCR Buffer and 50mM MgCl 2 )
DNase/RNase-Free Distilled Water
Shrimp Alkaline Phosphatase (1U/?L) USB Cat No. 70092Y
Exonuclease I(10U/?L) USB Cat No. 70073Z
Streptavidin R-Phycoerythrin (1mg/mL) Molecular Probes Cat. No. S-866

Equipment and Consumables Required

Equipment

LuminexR 100 xMAPT System
Manifold for vacuum pump system, such as the Qiagen QIAvac 96 (Cat # 19504)
Mini Centrifuge
Multichannel Pipette (150 ?L, 200 ?L)
Pipettes (P10, P200, P1000)
Racks for 1.5 mL
Racks for 0.2 mL thin wall tubes for PCR
Sonicator bath
Thermocycler for 0.2 mL thin wall PCR tubes and 96well plates
Vortex
Vacuum pump system

Consumables

Costar ThermowellT Thin-wall polycarbonate 96-well plates (Corning Cat No. 6509)
Costar Thermowell Aluminum Sealer ( Corning Cat No.6570)
0.2 mL thin wall tubes appropriate for Thermocycler
0.5 mL microcentrifuge tubes
1.5 mL microcentrifuge tubes
Multiscreen filter plates (MABV N12, Millipore)
Sealing mats to cover 96-well plate
Adhesive Sealing Film for Multiscreen filter plates
Aerosol resistant tips for pipettes
Reservoir basins

Introduction

The β -Thal Check 17 Kit simultaneously screens for the17 mutations of β -globin gene listed below:

Table 1

-101 C a T	cd8 -AA	cd39 C a T	3'UTR1570 T a C
-87 C a G	IVSI-1 G a A	IVSII-1 G a A	poly-b-A A a G
cd5 -CT	IVSI-5 G a A	IVSII-745 C a G
cd6 -A	IVSI-6 T a C	IVSII-848 C a A
HbS A a T	IVSI-110 G a A	3'UTR1480 C a G

The β -Thal Check 17 Kit incorporates duplex Polymerase Chain Reaction (PCR) and multiplex Allele Specific Primer Extension (ASPE) with Tm's proprietary Universal Tag sorting system on the LuminexR 100 xMAPT platform.

Method Overview

For each sample, 50ng of genomic DNA is amplified in a single duplex 25?L PCR reaction. The amplimers generated are 525bp and 750bp long.
To enable efficient incorporation of biotin-dCTP during the Allele Specific Primer Extension (ASPE) reaction each PCR product is treated with Shrimp Alkaline Phosphatase(SAP) and ExonucleaseI (EXO)to inactivate the nucleotides and to degrade the primers left over from the PCR reaction. An aliquot of 5?L from each treated PCR reaction is used in the ASPE reaction containing 31universally-tagged primers. The ASPE products are then sorted by hybridization to the bead mix and then incubated with streptavidin, R-phycoerythrin conjugate (reporter solution). Samples are read on the LUMINEX100 xMAP Instrument and signal is generated for each of the 16 mutations and their corresponding wild-type alleles. For each sample, these fluorescent values are then imported into Excel and analyzed to determine whether the wild-type and/or the mutant allele for each SNP are detected.

Sample preparation

It is recommended that the genomic DNA sample be extracted from whole blood using any method that yields genomic DNA with ratio of Optical Densities at 260 and 280nm (A 260 /A 280 ) higher than 1,8 and no apparent DNA degradation present, as seen on a 1% agarose gel electrophoresis. The concentration of extracted DNA should be adjusted to 5ng/?L (OD 260 = 0.1 Absorbance Unit per cm) in DNase-Free distilled water. A total of 50ng of extracted DNA is required for the 25?L PCR reaction. The DNA should be stored at 2-8°C until ready for use.

Software

The template required for running the samples on the LuminexR 100 xMAPT Instrument must be present on the computer that controls the LuminexR 100 xMAPT system.
Instructions for creating the template can be found in Section F.

Procedure

A. Duplex PCR Setup

The following procedure is for as many samples as it is required. Always include one negative control and prepare it last.

1. Thaw and bring to room temperature the β -globin PCR Primer Mix containing 4 PCR primers at a concentration of 5pmole/?L each
2. Vortex the PCR Primer Mix for 2-5secs and centrifuge briefly to bring the reagents to the bottom of the tube
3. Thaw the HotStart Master Mix of QIAGEN according to the instructions of the manufacturer *
4. Vortex the HotStart Master Mix for 2-5secs, centrifuge briefly to bring the reagents to the bottom of the tube and keep on ice until ready to use
5. Label the 0.2mL thin wall PCR tubes required with the names of the samples as well as a last tube as negative
6. Dispense in each tube 12,5?L HotStart Master Mix
7. Add 2,5?L β -globin PCR Primer Mix in every tube
8. Add 10?L genomic DNA sample (50ng) in the corresponding tube
9. In the last (negative control) tube add 10?L DNase-free, sterile ddH2O
10. Close the lids and centrifuge briefly to bring reagents to the bottom of the tubes
11. Place the tubes in the Thermocycler and run the cycling program described below
12. Store PCR products at 2-8oC until ready to use

Cycling Program :

Thermocycler temperature should be set as BLOCK temprature and the heated lid should be ON and set at 99o C.

1 cycle 95o C 15mins
35 cycles 95o C 30secs
60o C 30secs
72o C 1min
1 cycle 72o C 10mins
Hold at 4o C

B. Amplicon quality control

Prepare a 1.5%agarose gel in 1XTAE and run 5?L of the PCR reaction to verify successful amplification of both PCR products.
The amplicon sizes should be 525bp and 750bp and they should be reconfirmed with simultaneous run of appropriate DNA molecular weight marker.

C. Amplicon treatment

Vortex PCR tubes for 2-5secs and centrifuge briefly to bring samples to the bottom of the tube
In a 1,5ml tube mix Shrimp Alkaline Phosphatase and Exonuclease I at a ratio 2,5?L Shrimp Alkaline Phosphatase : 1?L Exonuclease I
Vortex enzyme mix for 2-5secs and centrifuge briefly to bring reagents to the bottom of the tube
Add 3.5?L of Exo/Sap enzyme Mix in every PCR tube containing 20?L PCR reaction
Vortex tubes for 2-5secs and centrifuge briefly to bring reagents to the bottom of the tube
Incubate tubes in a Thermocycler programmed as follows: 37 o C 30mins - 90 o C 15mins - 4o C HOLD
Store treated reactions at 2-8oC until ready to use

D. Multiplex ASPE

Thaw and bring to room temperature the tube of β -Thal 17 ASPE Primer Mix, the 10XASPE Buffer and 50mM MgCl 2 supplied with the Platinum Tsp DNA polymerase
Prepare a mix of dATP, dGTP and dTTP at a concentration of 0,1mM each
Vortex tubes for 2-5secs to mix reagents and centrifuge briefly to bring reagents to the bottom of the tube
Label the 0.2mL thin wall PCR tubes required with names corresponding to the PCR tubes
Label one 1.5ml tube as AMM and add the reagents listed below to prepare the ASPE Master Mix. The volumes listed correspond to one ASPE reaction. Calculate the total volumes by multiplying the numbers listed on the column 'Volume per reaction' by the number of PCR reactions. For 8 PCR reactions use the volumes listed in the column 'Volume for 8 reactions'

Reagents	Volume per reaction	Volume for 8 reactions
DNase-free water	10,1 ?L	80,8 ?L
10XASPEBuffer	2 ?L	16 ?L
50mM MgCl 2	0,5 ?L	4 ?L
dATP,dGTPanddTTP mix	1 ?L	8 ?L
dCTP-biotin	0,25 ?L	2 ?L
β -Thal 17 ASPE Primer Mix	1 ?L	8 ?L
Platinum Tsp DNA Polymerase	0,15 ?L	1,2 ?L
Total Volume	15 ?L	120

6. Vortex ASPE Master Mix for 2-5 seconds and centrifuge (2-5 seconds) to bring reagents to the bottom of tube.
7. Aliquot 15?L of the ASPE Master Mix into each of the 0.2mL ASPE tubes
8. Vortex tubes of treated PCR amlicons for 2-5 seconds to mix and centrifuge (2-5 seconds) to bring reagents to the bottom of tube.
9. Add 5 ?L of treated PCR product to correspondingly labelled tube containing 15 ?L ASPE Master Mix and cap it immediately after addition of sample
10. Vortex tubes for 2-5 seconds to mix and centrifuge (2-5 seconds) to bring reagents to the bottom of tube.
11. Place tubes in the Thermocycler and run the following cycling program:

Thermocycler temperature should be set as BLOCK temprature and the heated lid should be ON and set at 99o C

1 cycle 96o C 2min
40cycles 95o C 30secs
50o C 30secs
74o C 60secs
HOLD at 4o C

Store the ASPE reactions at 2-8 o C until ready to use

E. Bead Hybridization

NOTES:

Preparation of the Bead Mix (optional):
a. Prepare mix of beads enough for all the ASPE reactions as well as an additional No DNA control
b. Vortex the FlexMap Bead stock vials thoroughly
c. Use 2500 beads from each set per reaction
d. Mix all the FlexMap Bead sets together in one or more 1,5mL tubes to prepare the β -thal 17 FlexMap Bead Mix
e. Centrifuge at >8000g for 1-2mins
f. Discard liquid supernatant
g. Resuspend the Beads in 2x Tm Hybridization Buffer at a concentration of 100 beads of each set per ?L
Cut and label one or more 8-well strips from a 96-well PCR plate.
Vortex the β -thal 17 FlexMap Bead Mix tube for 10 seconds and then sonicate for 10 seconds to disperse the beads.
Repeat step3.
Dispense 15 ? L DNase-free H 2 O into the wells of the strips of 96-well plate
Aliquot 25 ? L of the β -thal 17 FlexMap Bead Mix into the wells of the strips of 96-well plate
Vortex the tubes of ASPE reactions (carried out in Section C: Multiplex ASPE) for 2-5 seconds and centrifuge for 2-5 seconds to bring reagents to the bottom of the tube.
Aliquot 10 ? L of each ASPE reaction into the correspondingly labelled well.
Add 10 ? L H 2 O in the well labelled as No DNA
Mix reagents by pipetting up and down several times with a 8-channel pipette
Cover the 8 well strip(s) cut from the 96-well plate with a sealing mat, making sure that all the wells are covered.
Place tubes in a Thermocycler programmed as follows:

96°C - 2 min, 37°C - 1 hour

NOTE: STEP 16 SHOULD BE STARTED WITHIN 5 MINUTES OF THE COMPLETION OF THE 1 HOUR INCUBATION AT 37°C .

13. Label the wells on a MultiScreen filter plate (Millipore) according to the labelling of the bead hybridization samples. Every 8 wells should be on the same column of the filter plate (A to H). For testing 16 samples, use two columns, and if testing 24 samples, use 3 columns.
14. Cover all wells not to be used with Adhesive Sealing Film or tape (for possible later use of the unused wells. Sealing of the unused wells also provides a better vacuum for the filtration steps).
15. Prepare 30ml 1XTmHybridization Buffer by diluting 2XTmHybridization Buffer with equal volume DNase-RNase free sterile H 2 O. Mix by vortex.
16. Before proceeding to the next step (about 5 minutes before completion of the 1 hour incubation in step12) prepare the reporter solution. Vortex the tube of Streptavidin, R-Phycoerythrin (SA-PE) conjugate, for 2-5 seconds.

For 8 samples , add 1.5 ? L of SA-PE (1mg/mL) to 0.75mL of 1X Tm Hybridization Buffer in a 1.5ml tube.
For 16 samples , add 3 ? L of SA-PE (1mg/mL) to 1.5mL of 1X Tm Hybridization Buffer in a 1.5ml tube.
For 32 samples , add 6 ? L of SA-PE (1mg/mL) to 3mL of 1X Tm Hybridization Buffer in two 1.5ml tubes.
Vortex for 10 seconds to mix. Protect from light until ready to use.

17. Place filter plate on manifold attached to a vacuum system able to provide a vacuum of 200-400mbar.
18. Transfer 7.0mL or more of the 1X Tm Hybridization Buffer into a reservoir basin.
19. Using a multi-channel pipette pre-wet the filter membrane for the required number of wells by transferring 150?L of 1X Tm Hybridization Buffer being careful not to touch the bottom of the filter plate. Apply vacuum until all the buffer has been drawn through. Turn off the vacuum. Repeat this step using a second 150?L aliquot of 1X Tm Hybridization Buffer. 20. Using a multi-channel pipette, aliquot 100?L of 1X Tm Hybridization Buffer from the reservoir into the hybridized samples
21. Using the 8-channel pipette, transfer entire contents (150?L) of the hybridized samples and 1X Tm Hybridization Buffer (from step 20) into the appropriately labelled wells of the filter plate, being careful not to touch the bottom of the filter plate.
22. Apply vacuum to completely remove all liquid from the wells. Do NOT over-dry the filter membrane. Turn off vacuum. 23. Wash beads by adding 200?L of the 1X Tm Hybridization Buffer into the wells with an 8-channel pipette.
24. Apply vacuum to completely remove all liquid from the samples. Turn off the vacuum.
25. Remove plate from manifold on vacuum and place it on a clean paper towel to blot dry its bottom.
26. Transfer the reporter solution (prepared in step 16) into a reservoir basin and using an 8-channel pipette, add 70 ? L of the reporter solution into each well, being careful not to touch the bottom of the filter plate.
27. Resuspend the beads by pipetting up and down several times with the 8-channel pipette, always careful not to touch the bottom of the plate.
28. Transfer the bead suspensions in a clean piece of one or more 8-well strips from a 96-well PCR plate.
29. Incubate the plate at 37 ° C for 15 minutes.

NOTE: The incubation should be carried out in a dark place, since the beads are light sensitive.

F. Data Acquisition

Creating the 'ASPE β -globin17 Assay' Template on the LuminexR100 xMAPT computer

NOTE: If the Template ' ASPE β -globin17 Assay' is already installed on the computer that controls the LuminexR 100 xMAPT system on which the assay will be run, you can skip this subsection.

1. Access the computer that controls the LuminexR 100 xMAPT system on which the assay will be run.
2. Open the LUMINEX 100IS Software V2.2.329, SP1
3. Click on Acq.Detail button
4. On the window that appears click on Create Template button
5. In the 'Template Setup Wizard' dialog box fill the Template Name (' ASPE β -globin17 Assay') , pick as Template Type the Data Collection Only, Sample vol. 50uL , Sample Time Out 25secs and Doublet Discriminator 8000-13000
6. Click Next
7. Insert the following allele Names and Bead ID's

FlexMAP bead set

allele name

-87n

-87m

-101n

-101m

CD5n

CD5m

CD6n

CD6m

HbS

CD8n

CD8m

IVSI-1n

IVSI-1m

IVSI-5n

IVSI-5m

IVSI-6n

IVSI-6m

IVSI-110n

IVSI-110m

CD39n

CD39m

IVSII-1n

IVSII-1m

IVSII-745n

IVSII-745m

IVSII-848n

IVSII-848m

3'UTR1480n

3'UTR1480m

3'UTR1570n

3'UTR1570m

POLYAn

POLYAm

8. In the column of UNITS fill the first cell with "MFI" and click Apply units
9. In the column of Min Beads fill the first cell with "100" and click Apply units
10. Click Next
11. From the list of Available Commands pick the Prime command
12. Click on the right looking arrow and select the command Acquire Test Speciment from the list of Available Commands
9. Click Save and Close

Instrument Preparation and Data Acquisition

1. Turn on the LuminexR 100 xMAPT and prepare instrument to read the samples by following the procedures described in the LuminexR 100 xMAPT User Manual. Calibrate the system as described in the User Manual. This should be carried out in advance of the hybridization reaction. It is recommended that a wash step be carried out prior to the next step.
2. The sample arm vertical height needs to be adjusted to read samples from the 96-well PCR plate. In an empty 96-well PCR plate supported on the setup XY platform , place one alignment sphere into well A1.
3. Refer to the LuminexR 100 xMAPT User Manual Version 2.2 (page A-10 to A-11) on procedures to adjust the sample probe vertical height.
4. Ensure that the temperature on the Setup XY platform is disabled so that readings are carried out at ambient temperature.
5. Create a new batch by clicking on New Batch on the toolbar. Select the template ' ASPE β -globin17' and click Select .
6. Fill in the batch Name, Description and Operator
7. Add the number of samples to be read, in the box next to "Acquired Patient", push "apply" and insert the Sample ID's to the list provided .
8. Select between Save and Load to run the batch immediately or Save only for later use of the batch and click Finish
9. At the end of 15 minutes incubation of the samples (step 28, Section E), Eject the plate holder, place the piece of 96-well PCR plate on the plate holder and Retract holder. The first sample to be read should always be placed in the top left corner of the plate holder (position A1).
10. Begin reading samples by pressing Start .
11. After the last sample has been read, the batch is automatically saved in the folder MyBatches in the Local Disc (C:)
12. Remove the strips of 96-well PCR plate from the plate holder on the LuminexR 100 xMAPT System.
13. If the vertical arm height needs to be reset to its original position, refer to the LuminexR 100 xMAPT User Manual Version 2.2 on procedures to adjust the sample arm vertical height.
14. Wash and soak LuminexR 100 xMAPT System, following standard procedures in the LuminexR 100 xMAPT User Manual.

9. Πρωτόκολλο ανίχνευσης 10 μεταλλαγών του γονιδίου της α-σφαιρίνης

Reagents required

NOTE: Τ he following reagents are included in the kit.

α 1-globin PCR Primer Mix
α 2-globin PCR Primer Mix
α 1-Thal ASPE Primer Mix
α 2-Thal ASPE Primer Mix
α 1-Thal FlexMap Bead Mix
α 2-Thal FlexMap Bead Mix
2X Tm Hybridization Buffer (0.4M NaCl, 0.2M Tris pH8.0, 0.16%TritonX-100)
(TritonX-100, SIGMA T-9284)
HotStart Taq Master Mix, QIAGEN Cat. No.203445
dNTPs 100mM each
dCTP-biotin 0.4mM, Invitrogen Cat.No.19518-018
Platinum Tsp DNA Polymerase (5 U/?L) Invitrogen Cat. No.11448-024
(including 10X PCR Buffer and 50mM MgCl 2 )
DNase/RNase-Free Distilled Water
Shrimp Alkaline Phosphatase (1U/?L) USB Cat No. 70092Y
Exonuclease I(10U/?L) USB Cat No. 70073Z
Streptavidin R-Phycoerythrin (1mg/mL) Molecular Probes Cat. No. S-866

Equipment and Consumables Required

Equipment

Consumables

Introduction

The α 1-Thal Check Kit simultaneously screens for the 4 mutations of α 1 globin gene listed below:

Table 1

Hb Heraklion	del CD37
HbTaybe	del CD38 or 39
Hb Agia Sofia	del CD62
Hb Questembert	CD131 TCT-> CCT

Note: mutated alleles CD37 and CD38 cannot be separately detected. The ASPE primer for CD38 mutated allele detects also CD37 mutation.

The α 2-Thal Check Kit simultaneously screens for the 7 mutations of α 2 globin gene listed below:

Table 2

Hb Agrinio	CD29 T->C	Hb Saudi	AATAA A ->AATAA G
Hph	IVSI-1 (-5nts TGAGG)	polyA (nt817) (or polyG)	AAT A AA->AAT G AA
IVSI-116acceptor	GC A GGA->GC G GGA
Hb Icaria	CD142 T AA-> A AA
Hb Constant Spring	CD142 T AA-> C AA

The α 1-Thal Check and α 2-Thal Check Kits incorporate a Polymerase Chain Reaction (PCR) and a multiplex Allele Specific Primer Extension (ASPE) with Tm's proprietary Universal Tag sorting system on the LuminexR 100 xMAPT platform.

Method Overview

For each sample, 50ng of genomic DNA is amplified in each of two separate 25?L PCR reactions. The amplimers generated are 836bp for α 2 and 715bp for α 1 .
To enable efficient incorporation of biotin-dCTP during the Allele Specific Primer Extension (ASPE) reaction each PCR product is treated with Shrimp Alkaline Phosphatase(SAP) and ExonucleaseI (EXO)to inactivate the nucleotides and to degrade the primers left over from the PCR reaction. An aliquot of 5?L from each treated PCR reaction is used in the ASPE reaction containing the universally-tagged primers. The ASPE products are then sorted by hybridization to the bead mix and then incubated with streptavidin-R-phycoerythrin conjugate (reporter solution). Samples are read on the LUMINEX100 xMAP Instrument and signal is generated for each of the 13 mutations and their corresponding wild-type alleles. For each sample, these fluorescent values are then imported into Excel and analyzed to determine whether the wild-type and/or the mutant allele for each SNP are detected.

Sample preparation

Software

Procedure

A. PCR Setup

The following procedure is for as many samples as it is required. Always include one negative control and prepare it last.

1. Thaw and bring to room temperature the α 1-globin PCR Primer and α 2-globin PCR Primer Mixes containing 2 PCR primers each at a concentration of 5pmole/?L each in 50% DMSO
2 .Vortex the PCR Primer Mix for 2-5secs and centrifuge briefly to bring the reagents to the bottom of the tube
3. Thaw the HotStart Master Mix of QIAGEN according to the instructions of the manufacturer *
4. Vortex the HotStart Master Mix for 2-5secs, centrifuge briefly to bring the reagents to the bottom of the tube and keep on ice until ready to use
5. Label the 0.2mL thin wall PCR tubes required with the names of the samples as well as a last tube as negative. Use separate tubes for the amplification of α 1 and α 2 globin genes
6. Dispense in each tube 12,5?L HotStart Master Mix
7. Add 2,5?L α 1 or α 2-globin PCR Primer Mix in every tube
8. Add 10?L genomic DNA sample (50ng) in the corresponding tube
9. In the last (negative control) tube add 10?L DNase-free, sterile ddH2O
10. Close the lids, vortex for 2-5secs and centrifuge briefly to bring reagents to the bottom of the tubes
11. Place the tubes in the Thermocycler and run the cycling program described below
12. Store PCR products at 2-8 o C until ready to use

Cycling Program :

Thermocycler temperature should be set as BLOCK temprature and the heated lid should be ON and set at 99o C.

1 cycle 95o C 10mins
35 cycles 96o C 30secs
60o C 45secs
72o C 1,5min
1 cycle 72o C 5mins
Hold at 4o C

B. Amplicon quality control

Prepare a 1.5%agarose gel in 1XTAE and run 5?L of each PCR reaction to verify successful amplification of both PCR products.
The amplicon sizes should be 836bp for α 2 and 715bp for α 1 and they should be reconfirmed with simultaneous run of appropriate DNA molecular weight marker.

C. Amplicon treatment

13. Vortex PCR tubes for 2-5secs and centrifuge briefly to bring samples to the bottom of the tube
14. The Exo/Sap enzyme Mix provided consists of Shrimp Alkaline Phosphatase and Exonuclease I at a ratio 2,5?L Shrimp Alkaline Phosphatase : 1?L Exonuclease I (2,5u SAP:10u ExoI)
15. Vortex enzyme mix for 2-5secs and centrifuge briefly to bring reagents to the bottom of the tube
16. Add 3.5?L of Exo/Sap enzyme Mix in every PCR tube containing 20?L PCR reaction
17. Vortex tubes for 2-5secs and centrifuge briefly to bring reagents to the bottom of the tube
18. Incubate tubes in a Thermocycler programmed as follows: 37o C 30mins - 90o C 15mins - 4o C HOLD
19. Store treated reactions at 2-8oC until ready to use

D. Multiplex ASPE

20. Thaw and bring to room temperature the tube of α 1- and α 2-Thal ASPE Primer Mixes, the 10XASPE Buffer and 50mM MgCl 2
21. Thaw the mix of dATP, dGTP and dTTP (0,1mM each) and keep on ice
22. Vortex tubes for 2-5secs to mix reagents and centrifuge briefly to bring reagents to the bottom of the tube
23. Label the 0.2mL thin wall PCR tubes required with names corresponding to the PCR tubes
24. Label two 1.5ml tubes as α 1 - AMM and α 2 - AMM and add the reagents listed below to prepare two separate ASPE Master Mixes for the α 1 and α 2 globin using the α 1- or α 2-Thal ASPE Primer Mixes respectively. The volumes listed correspond to one and eight ASPE reactions. Adjust the volumes to your number of reactions by multiplying the numbers listed on the column 'Volume per reaction' by the number of your PCR reactions.

Reagents	Volume per reaction	Volume for 8 reactions
DNase-free water	10,1 ?L	80,8 ?L
10XASPE Buffer	2 ?L	16 ?L
50mM MgCl 2	0,5 ?L	4 ?L
dATP,dGTPanddTTP mix	1 ?L	8 ?L
dCTP-biotin	0,25 ?L	2 ?L
α 1 or α 2 - Thal ASPE Primer Mix	1 ?L	8 ?L
Platinum Tsp DNA Polymerase	0,15 ?L	1,2 ?L
Total Volume	15 ?L	120

25. Vortex the ASPE Master Mixes for 2-5 seconds and centrifuge (2-5 seconds) to bring reagents to the bottom of tube.
26. Aliquot 15?L of each ASPE Master Mix into each of the 0.2mL accordingly labelled ASPE tubes
27. Vortex tubes of treated PCR amlicons for 2-5 seconds to mix and centrifuge (2-5 seconds) to bring reagents to the bottom of tube.
28. Add 5 ?L of treated PCR product to correspondingly labelled tube containing 15 ?L ASPE Master Mix and cap it immediately after addition of sample
29. Vortex tubes for 2-5 seconds to mix and centrifuge (2-5 seconds) to bring reagents to the bottom of tube.
30. Place tubes in the Thermocycler and run the following cycling program:

Thermocycler temperature should be set as BLOCK temperature and the heated lid should be ON and set at 99 o C

1 cycle 96o C 2min
40cycles 96o C 60secs
58o C 30secs
74o C 60secs
HOLD at 4o C

Store the ASPE reactions at 2-8o C until ready to use

E. Bead Hybridization

NOTES:

1. PRIOR TO THE HYBRIDIZATION REACTION, TURN ON THE LUMINEXR 100 xMAPT SYSTEM AND PREPARE INSTRUMENT TO READ THE SAMPLES BY FOLLOWING THE PROCEDURES DESCRIBED IN THE LUMINEXR 100 xMAPT USER MANUAL.
2. SINCE THE BEADS ARE LIGHT SENSITIVE LIMIT THEIR EXPOSURE TO LIGHT AT ALL TIMES DURING THE SETUP OF THE HYBRIDIZATION REACTION.

31. Preparation of the Bead Mix (optional):
a. Prepare mix of beads enough for all the ASPE reactions as well as an additional No DNA control
b. Vortex the FlexMap Bead stock vials thoroughly
c. Use 2500 beads from each set per reaction
d. Mix all the FlexMap Bead sets together in one or more 1,5mL tubes to prepare the α 1- and α 2-Thal FlexMap Bead Mixes
e. Centrifuge at >8000g for 1-2mins
f. Discard liquid supernatant
g. Resuspend the Beads in 2x Tm Hybridization Buffer at a concentration of 100 beads of each set per ?L
32. Cut and label one or more 8-well strips from a 96-well PCR plate.
33. Vortex every FlexMap Bead Mix tube for 10 seconds and then sonicate for 10 seconds to disperse the beads.
34. Repeat step3.
35. Dispense 15 ? L DNase-free H 2 O into the wells of the strips of 96-well plate
36. Aliquot 25 ? L of the α 1-thal FlexMap Bead Mix into the wells of the strips of a 96-well plate and the α 2-thal FlexMap Bead Mix into the wells of some other strips of a 96-well plate.
37. Vortex the tubes of ASPE reactions (carried out in Section C: Multiplex ASPE) for 2-5 seconds and centrifuge for 2-5 seconds to bring reagents to the bottom of the tube.
38. Aliquot 10 ?L of each ASPE reaction into the correspondingly labelled well.
39. Add 10 ?L H 2 O in the well labelled as No DNA
40. Mix reagents by pipetting up and down several times with a 8-channel pipette
41. Cover the 8 well strip(s) cut from the 96-well plate with a sealing mat, making sure that all the wells are covered.
42. Place tubes in a Thermocycler programmed as follows:

96°C - 2 min, 37°C - 1 hour

NOTE: STEP 16 SHOULD BE STARTED WITHIN 5 MINUTES OF THE COMPLETION OF THE 1 HOUR INCUBATION AT 37°C .

43. Label the wells on a MultiScreen filter plate (Millipore) according to the labelling of the bead hybridization samples. Every 8 wells should be on the same column of the filter plate (A to H). For testing 16 samples, use two columns, and if testing 24 samples, use 3 columns.
44. Cover all wells not to be used with Adhesive Sealing Film or tape (for possible later use of the unused wells. Sealing of the unused wells also provides a better vacuum for the filtration steps).
45. Prepare 30ml 1XTmHybridization Buffer by diluting 2XTmHybridization Buffer with equal volume DNase-RNase free sterile H2O. Mix by vortex.
46. Before proceeding to the next step (about 5 minutes before completion of the 1 hour incubation in step12) prepare the reporter solution. Vortex the tube of Streptavidin, R-Phycoerythrin (SA-PE) conjugate, for 2-5 seconds.

For 8 samples , add 1.5 ? L of SA-PE (1mg/mL) to 0.75mL of 1X Tm Hybridization Buffer in a 1.5ml tube.
For 16 samples , add 3 ? L of SA-PE (1mg/mL) to 1.5mL of 1X Tm Hybridization Buffer in a 1.5ml tube.
For 32 samples , add 6 ? L of SA-PE (1mg/mL) to 3mL of 1X Tm Hybridization Buffer in two 1.5ml tubes.
Vortex for 10 seconds to mix. Protect from light until ready to use.

47. Place filter plate on manifold attached to a vacuum system able to provide a vacuum of 200-400mbar.
48. Transfer 7.0mL or more of the 1X Tm Hybridization Buffer into a reservoir basin.
49. Using a multi-channel pipette pre-wet the filter membrane for the required number of wells by transferring 150?L of 1X Tm Hybridization Buffer being careful not to touch the bottom of the filter plate. Apply vacuum until all the buffer has been drawn through. Turn off the vacuum. Repeat this step using a second 150?L aliquot of 1X Tm Hybridization Buffer.
50. Using a multi-channel pipette, aliquot 100?L of 1X Tm Hybridization Buffer from the reservoir into the hybridized samples
51. Using the 8-channel pipette, transfer entire contents (150?L) of the hybridized samples and 1X Tm Hybridization Buffer (from step 20) into the appropriately labelled wells of the filter plate, being careful not to touch the bottom of the filter plate.
52. Apply vacuum to completely remove all liquid from the wells. Do NOT over-dry the filter membrane. Turn off vacuum.
53. Wash beads by adding 200?L of the 1X Tm Hybridization Buffer into the wells with an 8-channel pipette.
54. Apply vacuum to completely remove all liquid from the samples. Turn off the vacuum.
55. Remove plate from manifold on vacuum and place it on a clean paper towel to blot dry its bottom.
56. Transfer the reporter solution (prepared in step 16) into a reservoir basin and using an 8-channel pipette, add 70 ? L of the reporter solution into each well, being careful not to touch the bottom of the filter plate.
57. Resuspend the beads by pipetting up and down several times with the 8-channel pipette, always careful not to touch the bottom of the plate.
58. Transfer the bead suspensions in a clean piece of one or more 8-well strips from a 96-well PCR plate.
59. Incubate the plate at 37 ° C for 15 minutes.

NOTE: The incubation should be carried out in a dark place, since the beads are light sensitive.

F. Data Acquisition

Creating the 'ASPE α -globin Assay' Template on the LuminexR100 xMAPT computer

NOTE: If the Template ' ASPE α -globin Assay' is already installed on the computer that controls the LuminexR 100 xMAPT system on which the assay will be run, you can skip this subsection.

1. Access the computer that controls the LuminexR 100 xMAPT system on which the assay will be run.
2. Open the LUMINEX 100IS Software V2.2.329, SP1
3. Click on Acq.Detail button
4. On the window that appears click on Create Template button
5. In the 'Template Setup Wizard' dialog box fill the Template Name (' ASPE α -globin Assay') , pick as Template Type the Data Collection Only, Sample vol. 50uL , Sample Time Out 25secs and Doublet Discriminator 8000-13000
6. Click Next
7. Insert the following allele Names and Bead ID's

FlexMAP bead set

allele name

α2 :

α1 :

AGRn

AGRm

Hphn

Hphm

IVSI-116n

IVSI-116m

ICARIAn

ICARIAm

ConstantSpringm

polyA(817)n

polyA(817)m (GAA)

polyAn

Saudi polyAm (AAG)

TaybeCD38n

TaybeCD38m or Herakio CD37

AgiaSofiaCD62n

AgiaSofia CD62m

Questembert CD131n

Questembert CD131m

Instrument Preparation and Data Acquisition

1. Turn on the LuminexR 100 xMAPT and prepare instrument to read the samples by following the procedures described in the LuminexR 100 xMAPT User Manual. Calibrate the system as described in the User Manual. This should be carried out in advance of the hybridization reaction. It is recommended that a wash step be carried out prior to the next step.
2. The sample arm vertical height needs to be adjusted to read samples from the 96-well PCR plate. In an empty 96-well PCR plate supported on the setup XY platform , place one alignment sphere into well A1.
3. Refer to the LuminexR 100 xMAPT User Manual Version 2.2 (page A-10 to A-11) on procedures to adjust the sample probe vertical height.
4. Ensure that the temperature on the Setup XY platform is disabled so that readings are carried out at ambient temperature.
5. Create a new batch by clicking on New Batch on the toolbar. Select the template ' ASPE α -globin Assay' and click Select .
6. Fill in the batch Name, Description and Operator
7. Add the number of samples to be read, in the box next to "Acquired Patient", push "apply" and insert the Sample ID's to the list provided .
8. Select between Save and Load to run the batch immediately or Save only for later use of the batch and click Finish
9. At the end of 15 minutes incubation of the samples (step 28, Section E), Eject the plate holder, place the piece of 96-well PCR plate on the plate holder and Retract holder. The first sample to be read should always be placed in the top left corner of the plate holder (position A1).
10. Begin reading samples by pressing Start .
11. After the last sample has been read, the batch is automatically saved in the folder MyBatches in the Local Disc (C:)
12. Remove the strips of 96-well PCR plate from the plate holder on the LuminexR 100 xMAPT System.
13. If the vertical arm height needs to be reset to its original position, refer to the LuminexR 100 xMAPT User Manual Version 2.2 on procedures to adjust the sample arm vertical height.
14. Wash and soak LuminexR 100 xMAPT System, following standard procedures in the LuminexR 100 xMAPT User Manual.