Proceeding2562

1535 การประชุมวิชาการระดับชาติมหาวิทยาลัยทักษิณ ครั้งที่ 29 ประจ�ำปี 2562 วิจัยและนวัตกรรมเพื่อการพัฒนาที่ยั่งยืน Materials and methods Blood samples Forty RhD positive samples from Thai blood donor of regional blood centre XII Songkhla, Thailand were included in this study. The peripheral venous bloods from leftover blood specimen were collected in EDTA and CPDA1. Individual tube was divided into 3 microcentrifuge tubes with 1 ml and kept at 4 °C for DNA extraction at day 3, 10 and 30 after venipuncture. The use of leftover specimen was approved by the ethical committee of Faculty of Medicine, Prince of Songkla University (REC60-225-19-2). DNA preparation Genomic DNA (gDNA) was extracted from all samples by salting out technique (7). One milliliter of whole blood was centrifuged at 3,400 rpm for 5 min and 300 µl of buffy coat was transferred into fresh 12×75 mm glass tube. Red cells were lysed with 3 ml of RBC lysis buffer, mixed and centrifuged at 3,000 rpm for 10 min and supernatant was discarded. This step was repeated 3 times. The pellet was re- suspended in 1 ml RBC lysis buffer and transferred to new microcentrifuge tube. After centrifugation at 5,000 rpm for 2 min and supernatant was removed, 12 µl of proteinase K, 300 µl of ddH 2 O, 105 µl of 10% SDS and 105 µl of 7.5 M Guanidine HCl were added, mixed and incubated at 70 °C for 15 min. Supernatant was collected after centrifugation at 10,000 rpm for 10 min, then 1 ml of absolute ethanol was added and centrifuged. Supernatant was discarded and 469 µl of 80% ethanol was added, mixed for 1 minute and centrifuged at 10,000 rpm for 5 min. Incubated at 70 °C until dry. Diluted DNA with 100 µl of ddH 2 O and incubated at 70 °C for 5 min. The gDNA concentration was measured by NanoDrop Spectrophotometer (Thermo Scientific, USA) and kept at -20 °C for RhD genotyping by PCR-SSP. PCR amplification PCR-SSP was used to detect RHD gene using primer sets previously described elsewhere (12), forward primer 5’- ACG ATA CCC AGT TTG TCT -3’ and reverse primer 5’- TGA CCC TGA GAT GGC TGT -3’ (Integrated and technologies, Singapore) generated 2 amplicons; control fragment of 1200 bp and RHD specific fragment of 600 bp. Internal control is human growth primer sets, forward primer 5’- TGC CTT CCC AAC CAT TC CTT -3’ and reverse primer 5’- CCA CTC ACG GAT TTC TGT TGT GTT TC -3’ (Integrated and technologies) generated fragment of 429 bp. PCR mixture contained 4 µl of 5X HOT FIRE (Solis BioDyne), 0.2 µl of human growth forward and reverse primer, 0.5 µl of RHD forward and reverse primer. DNA concentration from each sample were normalized to 10 ng/  l, then 2 µl of DNA template was added into the mixture. PCR amplification was performed with initial activation at 95 °C for 15 min. Followed by 30 cycles at 95 °C for 30 s, 60 °C for 30 s, 72 °C for 45 s and final elongation at 72 °C for 5 min. PCR products were run on 2% agarose gel and stain by ethidium bromide. Then visualized with UV transluminator (UVITEC Cambridge) and compared with 100 bp DNA ladder. Due to various DNA concentration