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• W2892068288 abstract "When an embryo biopsy is performed prior to Preimplantation Genetic Testing for Aneuploidy (PGT-A), the biopsy is washed in PBS or similar buffer to remove residual culture media before being placed in a tube for Whole Genome Amplification (WGA). The amount of buffer transferred to the tube can vary from the recommended amount (<2 μL) to a larger volume. It is unknown what effect increased volumes of transfer buffer have on the WGA and subsequent PGT-A test result. This study aimed to determine the maximum volume of PBS that can be transferred into a tube with an embryo biopsy without compromising WGA using DOPlify. To replicate the effect of transferring different volumes of buffer with an embryo biopsy, a 5-cell equivalent amount of human genomic DNA (30pg, Promega) had incremental volumes of PBS (0-10 μL) added before standard DOPlify WGA (RHS Ltd). This was then repeated with 5-cell aliquots manually sorted from a 48,XXY,+21 cell line (GM04965, Coriell Institute) to determine the effect on cells representing a trophectoderm biopsy. Larger volume 40μL DOPlify reactions were also performed on 5-cell aliquots with an additional 0-20 μL of PBS added. WGA DNA was processed for PGT-A using the standard PG-Seq 48 sample protocol for MiSeq sequencing. Intensity of the WGA PCR product smear, DNA yield and karyotype after sequencing were analysed. A DNA smear was visualised for all genomic DNA 25 μL DOPlify amplifications with yields decreasing significantly (P<0.05) when 5 μL or more of PBS was added prior to the WGA reaction. A similar result was observed with the 5-cell aliquots, with the DNA yield decreasing as the volume of additional PBS increased; from 47 ng/μL with no PBS added to 8 ng/μL after 10μL of PBS was added. A significant decrease in yield (P<0.05) compared to the standard was observed after the addition of 6 μL of PBS. The correct karyotype results were obtained following the addition of 6 μL and 7 μL PBS, suggesting a reduction in WGA yield does not necessarily compromise the PGT-A test result. Expected PCR yields and correct karyotypes were obtained for the 40 μL master mix amplifications for all additional PBS volumes up to and including 8 μL however WGA yields dropped considerably for volumes higher than this. The efficiency of the WGA PCR is impacted by the volume of transfer buffer accompanying the embryo biopsy. Transfer buffer volumes of up to 5μL can be transferred into a tube with an embryo biopsy with no decrease in yield or adverse result after standard 25 μL DOPlify WGA. However, for transfer buffer volumes greater than 5 μL it is recommended that the WGA reaction volume is increased to 40 μL." @default.
• W2892068288 created "2018-09-27" @default.
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• W2892068288 date "2018-09-01" @default.
• W2892068288 modified "2023-10-16" @default.
• W2892068288 title "Embryo biopsy to tube transfer - how much PBS is too much?" @default.
• W2892068288 doi "https://doi.org/10.1016/j.fertnstert.2018.07.1179" @default.
• W2892068288 hasPublicationYear "2018" @default.
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