Fta elute micro cards

Three different commercially available Whatman^® cards with specific properties for sample collection, storage capacity, and costs were selected for comparative performance purposes: Whatman^® Classic Cards, FTA^® Elute Micro Cards, and 903 Protein Saver Cards (GE Healthcare Ltd., Cardiff, UK) (Table 1).
Whatman^® FTA^® Classic Cards contains chemical denaturants and a free radical scavenger that have the ability to lyse cells on contact, denature proteins, and protect DNA from degradation. The extracted DNA remains tightly bound to the matrix while cell membranes and organelles are lysed and proteins and inhibitors are washed away [26 ]. FTA^® Elute Micro Cards contains a chaotropic salt. Cells are lysed upon contact and proteins remain tightly bound while DNA is isolated from the matrix in a solution free of inhibitors with a simple water elution procedure [27 ]. Whatman^® 903 Protein saver Card is an untreated cotton fibber-based matrix. It does not stabilise nor protect DNA from degradation.

Gene loci at positions: IL-1A −889 (rs1800587), IL-1B +3954 (rs1143634), IL-4 −34 (rs2070874), IL-4 −590 (rs2243250), GATA-3 IVS4 +1468 (rs3802604) and COX-2 −1195 (rs689466) were analyzed using qPCR. 345 FTA elute micro cards (GE Healthcare UK, Little Chalfont, UK) from patients were used to obtain genomic DNA. A Ø 1.25 mm Harris micro punch (GE Healthcare UK, Little Chalfont, UK) was used to excise 6 pieces from a FTA card. The pieces were placed into a micro centrifuge tube, added with 500 μL of dH2O and vortexed 3x for 5 s. Thereafter, the pipette was used to remove the water. After 5 s centrifugation, the DNA elution was performed in 50 μL dH2O at 95 °C for 30 min in a heating block. The quality of DNA was analyzed by NanoDrop 2000 (Thermo Fisher Scientific, Watham/MA, USA) UV-Vis Spectrophotometer and monitored for the values of OD260/OD280. Then, 1 μL of the DNA extract was used as template in each qPCR. The qPCR was performed with the TaqMan SNP Assay^®, and the TaqMan Genotyping Master Mix^® (both Applied Biosystems Inc., Foster City/CA, USA) and analyzed using a Bio-Rad C1000 thermal cycler (Bio-Rad Laboratories Inc., Hercules/CA, USA). For validation of each SNP, one positive and two negative controls were included. Each sample was analyzed three times. The results of the TaqMan qPCR were verified by Sanger sequencing.