In order to determine 5-HTTLPR genotype, sterile swabs (Omni Swabs; Whatman) were used to obtain a buccal cell sample from each participant. Isolation of genomic DNA was performed using QIamp DNA Mini Kits from Qiagen. PCR protocol was followed for the subsequent genotyping (Glatz et al., 2003 (link)). Allelic variants were grouped into S’/S’ (S/S, S/Lg, Lg/ Lg) and L’/L’ (La/La). This bi-allelic classification is in line with previous studies (Markus and Firk, 2009 (link); Markus and De Raedt, 2011 (link); Markus and Capello, 2012 (link); Cerit et al., 2013 (link)).
Omni swabs
Manufactured by Cytiva
Sourced in United Kingdom
Omni Swabs are a versatile sampling device designed for collecting biological samples. They feature a soft, flexible tip and a sturdy handle, allowing for efficient and comfortable sample collection from various surfaces and locations.
Automatically generated - may contain errors
6 protocols using omni swabs
1
Buccal DNA Extraction and 5-HTTLPR Genotyping
2
Buccal, Blood, and Semen Sample Collection
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Buccal samples were collected using Omni Swabs (Whatman), which were scrapped along the inside cheek of healthy volunteers. Each sheet of FTA® paper contains four circular outlines printed directly on the paper as a guide to show where the biological sample should be added to the paper. For swab samples, the swab head was pressed directly onto the FTA® paper using three side-to-side motions to deposit the sample. Whole blood and semen samples were obtained from healthy volunteers. For liquid samples, a total of 40 µL was pipetted onto the printed circle area of the FTA® paper. During the analysis of semen samples, the use of 1 M dithiothreitol (DTT) (Sigma-Aldrich) was investigated as a reducing agent to increase sperm cell lysis. All samples were allowed to air dry for a minimum of 3 h before being placed in a desiccated storage environment until required.
3
Measuring HPA-1a Antibody Levels and Typing
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HPA-1a antibody (IgG) levels were measured using a modified MAIPA assay.[12 (link)]
DNA from the immunized women’s mothers was obtained from buccal swabs (Omni swabs, Whatman®, GE Healthcare UK Limited Buckinghamshire, UK). Purification of DNA was performed using a DNA isolation kit (QIAamp 96 Spin Blood kit, QIAGEN Inc., Valencia, CA, USA).
HPA-1 typing was performed using fluorogenic probes and a modified FAST 5´ Nuclease assay (NA)[13 (link)] or by flow cytometry.[14 (link)]
The HLA DRB3 typing was performed by sequencing the HLA DRB3 gene when present. For the PCR, we used intron-located amplification primers previously described by Kotsch et al.[15 (link)]
DNA from the immunized women’s mothers was obtained from buccal swabs (Omni swabs, Whatman®, GE Healthcare UK Limited Buckinghamshire, UK). Purification of DNA was performed using a DNA isolation kit (QIAamp 96 Spin Blood kit, QIAGEN Inc., Valencia, CA, USA).
HPA-1 typing was performed using fluorogenic probes and a modified FAST 5´ Nuclease assay (NA)[13 (link)] or by flow cytometry.[14 (link)]
The HLA DRB3 typing was performed by sequencing the HLA DRB3 gene when present. For the PCR, we used intron-located amplification primers previously described by Kotsch et al.[15 (link)]
4
Buccal Cell DNA Methylation Analysis
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DNA for methylation analysis was extracted from buccal cells, which were collected with OmniSwabs (Whatman®, kat. no. 28421853) from the children’s inner cheek. Subsequently, DNA was isolated with the QIAamp DNA Mini Kit (Qiagen, kat. no. 51306) according to the manufacturer’s protocol and stored at +4°C. 500 ng gDNA per sample was send to the Helmholtz-Zentrum München for genome-wide DNA methylation analysis. DNA methylation was analyzed using the Infinium Human Methylation 450K BeadChip array (Illumina).
5
Genetic Influences on Parenting Outcomes
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Our third and fourth aims are to test if the intervention effects on parenting or child outcomes are moderated by parental or children’s genotype or reactive temperament in line with a differential susceptibility model, [40 (link)]). Buccal cells will be collected from the children and both parents using Whatman Omniswabs in order to obtain information about genetic polymorphisms of specific dopamine related genes. Parental and children’s reactive temperament will be measured using subscales from Rothbarth’s temperament questionnaires (fearful and reactive temperament, sensory sensitivity; [34 (link), 41 (link)]).
6
Buccal DNA Extraction and 5-HTTLPR Genotyping
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Sterile swabs (Omni Swabs; Whatman, Maidstone, United Kingdom) were used to acquire
buccal cell samples for 5-HTTLPR determination. Isolation of genomic DNA was performed
using QIamp DNA Mini Kits (Qiagen, Leusden, The Netherlands). Polymerase chain reaction
protocol was followed for the subsequent 5-HTTLPR genotyping (Glatz et al., 2003 (link)). More recent findings revealed
that fragments in the 5-HTTLPR L-allele affect the transcription of the 5-HT transporter
including a high-expressive La and a low-expressive Lg variant
(Nakamura et al., 2000 (link)).
Consistent with previous research (Zalsman et al., 2006 (link)), tri-allelic variants were therefore reclassified into a
functionally relevant bi-allelic model including S’/S’ (S/S, S/Lg,
Lg/Lg), S’/L’ (S/La, Lg/La) and
L’/L’ (La/La) respectively. Hardy–Weinberg equilibrium (HWE) was
determined for the initial database and revealed that genotype frequencies of S’/S’
(n = 190), S’/L’ (n = 405) and L’/L’
(n = 209) did not differ from HWE (χ2 = 0.052,
p = 0.820).
buccal cell samples for 5-HTTLPR determination. Isolation of genomic DNA was performed
using QIamp DNA Mini Kits (Qiagen, Leusden, The Netherlands). Polymerase chain reaction
protocol was followed for the subsequent 5-HTTLPR genotyping (Glatz et al., 2003 (link)). More recent findings revealed
that fragments in the 5-HTTLPR L-allele affect the transcription of the 5-HT transporter
including a high-expressive La and a low-expressive Lg variant
(Nakamura et al., 2000 (link)).
Consistent with previous research (Zalsman et al., 2006 (link)), tri-allelic variants were therefore reclassified into a
functionally relevant bi-allelic model including S’/S’ (S/S, S/Lg,
Lg/Lg), S’/L’ (S/La, Lg/La) and
L’/L’ (La/La) respectively. Hardy–Weinberg equilibrium (HWE) was
determined for the initial database and revealed that genotype frequencies of S’/S’
(n = 190), S’/L’ (n = 405) and L’/L’
(n = 209) did not differ from HWE (χ2 = 0.052,
p = 0.820).
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