Dna rna mini kit

Bone marrow aspirates from newly diagnosed patients with multiple myeloma, entered into the UK MRC Myeloma IX study, were obtained after informed consent. PCs were selected using CD138 microbeads and magnet-assisted cell sorting (Miltenyi Biotec, Bergisch Gladbach, Germany) as described previously and PC purity was confirmed to be >90% in all cases by cytospin. RNA and DNA were extracted using commercially available kits (RNA/DNA mini kit or Allprep kit; QIAGEN) according to manufacturers' instructions.

Total RNA of tissues was extracted using TRIzol (Invitrogen) and RNA/DNA mini Kit (QIAGEN) according to the manufacturer′s protocol. Reverse transcription was carried out with PrimeScript RT reagent Kit with gDNA Eraser (TaKaRa) according to the manufacturer′s instructions. The cDNA was amplified by All‐in‐One™ qPCR Mix (GeneCopoeia, America) according to the manufacturer′s instructions in a SLAN‐96P system. The primers were designed by Primer 3.0 and primer sequence of each circRNA and β‐actin in this study were listed (Table 1).The conditions of amplification for hsa_circ_0005927 and hsa_circ_0069397: one cycle at 95°C for 10 minutes, 40 cycles at 95°C for 10 seconds followed by 61°C for 60 seconds; for hsa_circ_0001936: one cycle at 95°C for 10 minutes, 35 cycles at 95°C for 10 seconds followed by 64°C for 55 seconds; for hsa_circRNA_406010: one cycle at 95°C for 10 minutes, 40 cycles at 95°C for 10 seconds followed by 60°C for 45 seconds; for HECW2: one cycle at 95°C for 10 minutes, 40 cycles at 95°C for 10 seconds followed by 67°C for 45 seconds. Sequencing was applied to validate whether the products of qPCR were our target products. β‐actin served as internal control.

Total DNA of the human liver tissue samples were extracted by using the RNA/DNA Mini Kit (Qiagen, Hilden, Germany). DNA was diluted to working concentrations of 50 ng/μL for SNP chip genotyping. The genome-wide scan was performed using the Affymetrix Genome-Wide Human SNP Array 6.0. Quality control (QC) filtering of the GWAS data was performed by excluding arrays with a contrast QC<0.4 from further data analysis. The sex of each sample was determined using Genotyping Console, and none of them mismatched established and annotated sexes. Genotype data were generated using the birdseed algorithm.13 (link) SNPs were further filtered based on annotation, call rate, Hardy-Weinberg equilibrium, and allele frequency information. As a result, among the initially genotyped 909 622 SNPs on the Affymetrix Genome-Wide Human SNP Array 6.0 platform, 4023 duplicated SNPs and 1175 SNPs that did not have chromosomal annotation were first removed from further analysis. We then removed 54 656 SNPs that had a genotyping call rate <95%, and 737 SNPs that deviated significantly from Hardy-Weinberg equilibrium at the threshold (p<10⁻⁴). Considering the small sample size of our dataset, we removed a further 54 656 SNPs that had minor allele frequency (MAF) <25%. In total, 302 483 SNPs remained for further analysis.