TUBING FOR GENETIC ANALYSIS AFTER EMBRYO BIOPSY E. Velilla García, Phd Center for Embryo Medicine Director
CEM · Capità Arenas, 3-5, local 2 · 08034 Barcelona · Spain · +34 93 280 40 28 ·
[email protected] · www.pgdcem.com
KEY FACTORS
Good starting material Tubing protocol Monogenic diseases aCGH Avoiding contamination
CEM · Capità Arenas, 3-5, local 2 · 08034 Barcelona · Spain · +34 93 280 40 28 ·
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STARTING MATERIAL
Blastomere (D+3) A clear nucleus observed One biopsied cell per embryo − If cell lysis, biopsy of a second one
Trophectoderm (D+5) Around 5 cells sample
CEM · Capità Arenas, 3-5, local 2 · 08034 Barcelona · Spain · +34 93 280 40 28 ·
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TUBING PROTOCOL (I) Necessary material
Cold block
PCR plate & tubes
Pipette
Filtered tips
Specific for monogenic diseases
Washing Buffer (PBS 0,1 % PVA)
Lysis buffer
Mineral oil
Marker
80-140 μm pipettes
Petri dishes
Specific for aCGH
PBS 1% PVP
CEM · Capità Arenas, 3-5, local 2 · 08034 Barcelona · Spain · +34 93 280 40 28 ·
[email protected] · www.pgdcem.com
TUBING PROTOCOL (I) Necessary material
CEM · Capità Arenas, 3-5, local 2 · 08034 Barcelona · Spain · +34 93 280 40 28 ·
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TUBING PROTOCOL (II) MONOGENIC DISEASES: Washing dish Washing Buffer drops for blastomere washing (20l)
Washing Buffer drops for pipette washing between blastomere washing steps (40 μl) CEM · Capità Arenas, 3-5, local 2 · 08034 Barcelona · Spain · +34 93 280 40 28 ·
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TUBING PROTOCOL (II) MONOGENIC DISEASES: Tubing protocol 1
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CEM · Capità Arenas, 3-5, local 2 · 08034 Barcelona · Spain · +34 93 280 40 28 ·
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TUBING PROTOCOL (II) MONOGENIC DISEASES: Tubing protocol
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8 BLASTOMERE + 5 µl lysis buffer + one drop mineral oil Label tube with the corresponding #
CEM · Capità Arenas, 3-5, local 2 · 08034 Barcelona · Spain · +34 93 280 40 28 ·
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TUBING PROTOCOL (II) MONOGENIC DISEASES: Tubing protocol
9
NEGATIVE CONTROL Medium from the last washing drop + 5 µl lysis buffer + one drop mineral oil Label tube with the corresponding #
CEM · Capità Arenas, 3-5, local 2 · 08034 Barcelona · Spain · +34 93 280 40 28 ·
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TUBING PROTOCOL (III) aCGH: Washing dish
PBS-1%PVP drops for blastomere washing (20l)
PBS-1%PVP drops for pipette washing between blastomere washing steps (20 μl)
CEM · Capità Arenas, 3-5, local 2 · 08034 Barcelona · Spain · +34 93 280 40 28 ·
[email protected] · www.pgdcem.com
TUBING PROTOCOL (III)
aCGH: Tubing protocol Similar procedure of washing steps as in monogenic diseases Cell tubing in 2 µl of PBS-1%PVP Total volume 2.5 µl
No adding of mineral oil A negative control for each sample, taken from the last washing drop
CEM · Capità Arenas, 3-5, local 2 · 08034 Barcelona · Spain · +34 93 280 40 28 ·
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TUBING PROTOCOL (III) aCGH: Tubing protocol
1.Washing dish preparation
CEM · Capità Arenas, 3-5, local 2 · 08034 Barcelona · Spain · +34 93 280 40 28 ·
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TUBING PROTOCOL (III) aCGH: Tubing protocol
2.PCR tubes preparation CEM · Capità Arenas, 3-5, local 2 · 08034 Barcelona · Spain · +34 93 280 40 28 ·
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TUBING PROTOCOL (III) aCGH: Tubing protocol
3.Aspirating system preparation CEM · Capità Arenas, 3-5, local 2 · 08034 Barcelona · Spain · +34 93 280 40 28 ·
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TUBING PROTOCOL (III) aCGH: Tubing protocol 1
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4.Washing of the blastomere
CEM · Capità Arenas, 3-5, local 2 · 08034 Barcelona · Spain · +34 93 280 40 28 ·
[email protected] · www.pgdcem.com
TUBING PROTOCOL (III) aCGH: Tubing protocol
5A.Tubing of the blastomere in a stereoscopic microscope
CEM · Capità Arenas, 3-5, local 2 · 08034 Barcelona · Spain · +34 93 280 40 28 ·
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TUBING PROTOCOL (III) aCGH: Tubing protocol
5B.Tubing of the blastomere in a stereoscopic microscope
CEM · Capità Arenas, 3-5, local 2 · 08034 Barcelona · Spain · +34 93 280 40 28 ·
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AVOIDING CONTAMINATION Sources of contamination: Maternal
Exhaustive decumulation
Paternal
ICSI
External -Always use head cover, mask and gloves
-If possible, reserve a work place in IVF lab and clean it before work -Reserve a place in incubator -Maximum precaution along the tubing process CEM · Capità Arenas, 3-5, local 2 · 08034 Barcelona · Spain · +34 93 280 40 28 ·
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TUBING PROTOCOL (IV) General considerations: what to avoid
TOUCHING THE SKIN WITH CLEAN GLOBES CEM · Capità Arenas, 3-5, local 2 · 08034 Barcelona · Spain · +34 93 280 40 28 ·
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TUBING PROTOCOL (IV) General considerations: what to avoid
TALKING WITHOUT SURGICAL MASK CEM · Capità Arenas, 3-5, local 2 · 08034 Barcelona · Spain · +34 93 280 40 28 ·
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TUBING PROTOCOL (IV) General considerations: what to avoid
TOUCHING THE “DIRTY” END OF THE MOUTHPIPETTE CEM · Capità Arenas, 3-5, local 2 · 08034 Barcelona · Spain · +34 93 280 40 28 ·
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TUBING PROTOCOL (IV) General considerations: what to avoid
KEEPING THE TUBES OPENED CEM · Capità Arenas, 3-5, local 2 · 08034 Barcelona · Spain · +34 93 280 40 28 ·
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TUBING PROTOCOL (IV) General considerations: IVF lab, in laminar flow hood Wear appropriate lab suit Use of head cover and sterile clean gloves
Change gloves whenever necessary! Use of filtered pippete tips Handle PCR tubes carefully
Touch only the outside Keep closed as much as possible Avoid touching the “dirty” end of the mouthpipette
CEM · Capità Arenas, 3-5, local 2 · 08034 Barcelona · Spain · +34 93 280 40 28 ·
[email protected] · www.pgdcem.com
THANK YOU VERY MUCH FOR YOUR ATTENTION
CEM · Capità Arenas, 3-5, local 2 · 08034 Barcelona · Spain · +34 93 280 40 28 ·
[email protected] · www.pgdcem.com
