Rotor gene q pcr machine

An Allprep DNA/RNA mini kit (Qiagen, Hilden, Germany) was used to extract total RNA and genomic (g) DNA. From 1 μg of total RNA, complementary (c)DNA was synthesized using the QuantiTect Reverse Transcription Kit (Qiagen, Hilden, Germany) according to the manufacturer’s protocol. RT-PCR was performed at specific conditions using 1 μL of cDNA, specific primers (Table 1A), a Promega GoTaq^® qPCR Master mix (Promega, Madison, WI, USA), and a Qiagen Rotor-gene qPCR machine (Qiagen, Hilden, Germany). Expression levels of the target gene (CXCL1) were normalized to GAPDH expression as an internal control.
For bisulfite modification of the DNA, an aliquot of gDNA (2 μg) was treated with an EpiTect Bisulfite Kit (Qiagen, Hilden, Germany). MSP was conducted using 1 μL of the bisulfite-treated DNA, primers specifically designed for methylated and unmethylated DNA sequence of the promoter region of CXCL1 genes (Table 1B), a Promega GoTaq^® qPCR Master mix (Promega, Madison, WI, USA) and a Qiagen Rotor-gene qPCR machine (Qiagen, Hilden, Germany). Fully methylated and fully unmethylated control DNAs were purchased from Qiagen and were used as positive and negative controls, respectively. DNA methylation levels were calculated as described previously [36 (link)].

Total genomic (g)DNA was extracted from whole blood samples obtained from the healthy controls (n = 5) and COVID-19 patients (n = 5 for each asymptomatic and symptomatic) using QIAamp DNA Mini Kit mini kit (Qiagen, Hilden, Germany).
An aliquot gDNA (2 μg) was treated with EpiTect Bisulfite Kit (Qiagen). qMSP was conducted using 1 μl of the sodium bisulfite-treated DNA, primers specifically designed for methylated and unmethylated DNA sequence of the promoter region of HSPA1L and ULBP2 genes (Supplementary Table 1), Promega GoTaq^® qPCR Master Mix (Promega); and analyzed using Qiagen Rotor-gene qPCR machine (Qiagen). Based on the (cycle threshold) Ct values of methylated (M) primer amplification and un-methylated (U) primer amplification for a gene, a methylation level of the gene promoter was calculated as the fraction of M molecules in the total number of bisulfite-treated DNA molecules (Ct of M molecules/Ct of M molecules + Ct of U molecules) (Paschos and Allday, 2010 (link)). For each gene, methylation levels of asymptomatic COVID-19, as well as symptomatic COVID-19 were compared amongst healthy control. Fully methylated and fully unmethylated control DNAs were purchased from Qiagen and were used as positive and negative controls, respectively and to assess the behavior and specificity of the primers used in this study (Supplementary Figure 1).