Dna rna kit

BM mononuclear cells (MNC) were purified on Ficoll gradient and were processed for DNA extraction using the DNA/RNA Kit (Qiagen, Hilden, Germany). Mutations in SF3B1 were screened by Sanger sequencing or next generation sequencing of a panel of 37 genes (ASXL1, ATM, BCOR, BCORL1, BRAF, CBL, CEBPA, CUX1, DDX41, DNMT3A, EP300, ETV6, EZH2, FLT3, GATA2, IDH1, IDH2, JAK2, KIT, KRAS, NRAS, MPL, NPM1, PHF6, PTPN11, RAD21, RIT1, RUNX1, SETBP1, SF3B1, SRSF2, STAG2, TET2, TP53, U2AF1, WT1, ZRSR2).

Isolation of total RNA and cDNA synthesis were carried out as per manufacturer’s instructions. RNA was isolated from the SiHa cells using DNA/RNA kit (Qiagen Cat# 80204) and ~1 μg of RNA was transcribed into cDNA using MMLV reverse transcriptase (Promega Cat# M1701), 0.5 µl of dNTPs, 0.5 µl of RNase inhibitor, 4 µl of reverse random primer in a total volume of 20 μl. Specific transcripts were amplified using gene-specific primers:
HPV E6-
Forward: 5′-ATGCATGGAGATACACCTACATTG-3′
Reverse: 5′-CATTACATCCCGTACCCTCTTC-3′;
HPV E7-
Forward: 5′-ATGCACCAAAAGAGAACTGCAATGT-3′
Reverse: 5′-TTACAGCTGGGTTTCTCTACGTG-3′;
β-actin-
Forward: 5′-AGACTTCGAGCAGGAGATG-3′
Reverse: 5′-CTTGATCTTCATGGTGCTAGG-3′
Amplifications were carried out for 25 cycles on a Veriti Thermal cycler (Applied Biosystems) and the products were visualized by ethidium bromide staining after electrophoresis on 2% agarose gel. The bands corresponding to specific transcripts were scanned using a densitometer and normalized against the values corresponding to β-actin transcript bands.

Bone marrow mononuclear cells were purified on Ficoll gradients, and pellets were processed for nucleic acid extraction using a DNA/RNA Kit (Qiagen). Genomic DNA was studied by high-throughput sequencing of 45 genes recurrently mutated in myeloid malignancies using a panel designed on Human genome hg19, and sequencing was performed on Ion PGM™ (Life Technologies) on a dedicated 318 V2 chip [60 ]. Libraries were prepared using Ion AmpliSeq library kit2 384 (Life Technologies) according to the manufacturer’s instructions. The average coverage per gene was ≥ 500×. Reads were aligned against human genome build 19 (hg19) and analyzed for single nucleotide variant (SNV) calling with the NextGENe software (SoftGenetics, Chicago, IL) and with an in-house pipeline (Polydiag, Institut Imagine, Université de Paris). We reported all clinically relevant variants with a variant allele frequency (VAF) cutoff at 2%. All the samples were also screened for ASXL1 (including c.1934dupG; p.G646WfsX12) and SRSF2 mutations by Sanger sequencing. Moreover, aligned reads from .bam files were visualized using the Integrative Genomics Viewer v2.3 from the Broad Institute (Cambridge, MA, USA). Assessment of variant implication was performed based on population databases (dbSNP and GnomAD), mutation databases (COSMIC), and prediction software (Alamut, mutation taster, OncoKB, and Cancer Genome Interpreter).