Veriti pcr

165 SSR core set primers (Orjuela et al. 2010 (link)), which are evenly distributed across 12 chromosomes with a distance ranging between 2 and 3.5 Mbp were used to estimate the genetic relatedness among 23 genotypes. PCR reactions were carried out in Thermal cycler (Veriti PCR, Applied Biosystems, USA) with the total reaction volume of 10 μl containing 15 ng of genomic DNA, 1X assay buffer, 200 μM of dNTPs, 1.5 mM MgCl₂, 10 pmol of forward and reverse primer and 1 unit of Taq DNA polymerase (Thermo Scientific). PCR cycles were programmed as follows: initial denaturation at 94 °C for 5 min followed by 35 cycles of 94 °C for 45 s, 55 °C for 30 s, 72 °C for 45 s, and a final extension of 10 min at 72 °C. Amplified products were resolved on 4 % metaphor agarose gels prepared in 0.5 X TAE buffer and electrophoresis was conducted at 120 V for 2 h. Gels were stained with ethidium bromide and documented using gel documentation system (Alpha imager, USA). Only clear and unambiguous bands of SSR markers were scored. Amplified fragments were scored for the presence (1) or absence (0) of the corresponding band among the genotypes, and formed a data matrix comprising ‘1’ and ‘0’.

The extracted genomic DNA was amplified using Polymerase Chain Reaction (PCR). The primers used for amplification of FOXO3a gene are shown in Table 2 and were synthesized (Eurofins Genomics, Bangalore, India). PCR was performed on Veriti PCR (Applied Biosystems, USA). The PCR reaction comprised 20 μL volume with 0.5 μL of 100 ng DNA, 1 μL of 10 mM dNTPs (Bangalore Genei, India), 0.3 μL of 10 pmol of forward and reverse primer each, 0.3 μL of 3 U Taq DNA polymerase (Bangalore Genei, India), 3μL of Taq polymerase Buffer A (Bangalore Genei, India), and 14.6 μL of deionized water. The thermal cycling conditions were 5 minutes for initial denaturation at 95°C, 35 cycles at 95°C for 30 seconds for denaturation, 30 seconds at 62°C for annealing, and 1 minute at 72°C for extension, followed by 5 minutes at 72°C for final extension. The PCR products were separated on a 1.5% agarose gel containing 1.5 μL (stock of 10 mg/mL) ethidium bromide. A 100 bp DNA marker (Bangalore Genei, India) was used as a size standard for each gel lane. The gel was visualized under UV light using a gel documentation unit (Alpha Imager HP).

PCR reaction was standardized for exon 2, exon 3 and exon 4 of as described by Hviid et al. (18 (link)) in a Veriti PCR (Applied Biosystems, United States) to amplify the exonic regions of HLA-G gene using cDNA as the template. After optimization of PCR conditions, expression study of HLA-G isoforms was carried out in 80 HNSCC tissue samples and their adjacent normal tissues in a total volume of 15 μL using 2 μL of template cDNA, 1X PCR buffer, 1.5 mM MgCl₂, 200 μM of each deoxynucleotide triphosphate (dATP, dTTP, dCTP, dGTP), 0.5 μM of each primers and 1.5 U of Taq DNA polymerase. To determine whether the isoforms are membrane bound or soluble, PCR was performed using exon 5 specific primer sequences as described by Van and Ober (19 (link)). Isoforms of HLA-G were classified according to the presence the exon 2, exon 3, exon 4 and exon 5 as described by Amiot and Samson (20 (link)). Specificity of band amplification was determined by sequencing of 15% of the amplicons.