Allprep dna rna universal kit

KRAS mutation analysis was performed in the Genetics and Molecular Pathology Department at Monash Health, using the clinically validated KRAS StripAssay™ (ViennaLab Diagnostics) in accordance with manufacturer protocols and standard clinical practice. Where possible, DNA was extracted from fresh frozen EUS-FNA biopsies sourced from the multi-centre VPCB using the AllPrep DNA/RNA Universal Kit (Qiagen), although archival formalin-fixed paraffin embedded (FFPE) tissue was used where fresh frozen biopsy tissue was not available. The isolation of gDNA from FFPE tissue was performed on 5 x 10 micron-thick sections using the ReliaPrep FFPE gDNA Miniprep System (Promega). Prospective tissue testing on diagnostic biopsies was preferred, although archival or previously stored specimens from the VPCB could be used where fresh tissue was not feasible or available. DNA samples were quantified using the Qubit Fluorometer (Life Technologies) and quality assessed by TapeStation (Agilent Technologies). At the time of consent to sample collection for the VPCB, patients could elect for their treating physician to be contacted in the event of a significant genomic finding. KRAS wild-type results were notified to treating physicians who were able to offer referral for screening for the study if they deemed it clinically appropriate.

Fresh adenoma and adjacent healthy tissue samples were collected and snap-frozen immediately after resection from patients in the endoscopy program for the removal of large (> 1 cm) colorectal adenomas at the University Medical Centers Amsterdam (location AMC). All patients provided written informed consent (METC2015_206). mRNA was isolated from tissue samples by mechanical disruption using the FastPrep-24-5G (MP Biomedicals) in combination with Lysis Tubes S (Qiagen, Cat No./ID: 19091) for 2 times 1 min at 6.5/s with 1 min on ice in between followed by the Allprep DNA/RNA Universal kit (Qiagen, ID 80204).
For generation of human FAP organoids, adenomatous material from the colon was collected at the University Medical Centers Amsterdam (location AMC), after being approved by the Medical Ethical Committee (normal tissue: MEC 09/146 and MEC 05/071).

Total RNA and DNA was isolated using the QIAcube system with the AllPrep DNA/RNA Universal Kit (cat.no. 80224, Qiagen, Hilden, Germany) with 30 mg tissue as input. The tissue was manually minced with a scalpel on ice followed by homogenization using TissueLyzer LT and Qiashredder (Qiagen). RNA and DNA extraction was performed according to the protocol provided by the supplier. Nucleic acid concentrations were measured on a NanoDrop ND-1000 spectrophotometer (Thermo Scientific, Wilmington, DE, USA) and RNA integrity was analyzed using Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, USA).

Tissues were snap-frozen within 30 min after resection and stored at − 80 °C until the time of analysis. For nucleic acid isolation, 10 to 15 tissue cryosections (10 to 15 µm each) were prepared for each patient. The first and the last sections in each series were stained with hematoxylin and eosin (H&E) and tumor samples were reviewed by an experienced lung pathologist to determine the proportions of viable tumor cells, stromal cells, normal lung cell cells, infiltrating lymphocytes and necrotic areas. Only samples with a viable tumor content of ≥ 50% were used for subsequent analyses. Frozen tumor cryosections and matched normal lung tissue pieces were homogenized using the TissueLyser mixer-mill disruptor (2 × 2 min at 25 Hz, Qiagen, Hilden, Germany). Total DNA was isolated with the AllPrep DNA/RNA Universal Kit (Qiagen, Hilden, Germany) following the manufacturer’s instructions. DNA was stored at − 80 °C until further use.

We collected fresh paired biopsies from adenomas and the surrounding macroscopically normal‐appearing intestinal epithelium from 61 patients undergoing routine colectomy. The collection of material was approved by the medical ethical committee of the Amsterdam UMC under approval number (2015–206), and all patients provided written informed consent. Biopsies were dissociated using the FastPrep‐24‐5G (MP Biomedicals) in combination with Lysis Tubes S (Qiagen) for two times 1 min at 6.5/s with 1 min on ice in between, material was collected using the Allprep DNA/RNA Universal kit (Qiagen). The isolation and maintenance of human colon organoids were described before (van Neerven et al, 2021 (link)). Collection of material was approved by the Medical Ethical Committee of the Amsterdam AMC under approval numbers 2014–178 and 09–146 with written informed consent of the patients, and the experiments conformed to the principles set out in the WMA Declaration of Helsinki and the Department of Health and Human Services Belmont Report.

RNA was extracted from cell and tissue samples using AllPrep DNA/RNA Universal Kit according to the manufacturer’s instructions (Qiagen). cDNA was generated from RNA using a High- Capacity RNA to cDNA Kit (Applied Biosystems).
Each RT-PCR reaction utilized 10 ng cDNA and was analyzed in technical duplicates. Reactions were analyzed on a 7500 Fast Real-Time PCR System (Applied Biosystems). All primer/probes used in this study utilized the FAM–MGB TaqMan system and were purchased from Applied Biosystems or were generated using the primer design tool Primer-BLAST (http://www.ncbi.nlm.nih.gov/ tools/primer-blast) (Additional file 10: Table I). Amplification of GAPDH was used as an internal standard. The Ct method was used to determine the fold-difference in expression levels relative to a control sample.

DNA isolation for exome sequencing was carried out at Theragen Etex Bio Institute (Seoul, South Korea). DNA was isolated using QIAamp DNA Mini Kit (Qiagen cat.# 51306) per the manufacturer’s protocol. DNA from two samples (S159_14_11 and S176_14_11) was isolated using CTAB Extraction Solution (Biosesang cat.# C2007) per the manufacturer’s protocol. DNA integrity was assessed by electrophoresis, and concentration was determined using the Nanodrop ND-1000 spectrophotometer (Thermo Scientific cat.# ND-1000) and Qubit fluorometer (Thermo Scientific cat.# Q33226). Total RNA and DNA isolation for gene expression microarrays was carried out using the QIAcube system (Qiagen cat.# 9001292) with the AllPrep DNA/RNA Universal Kit (Qiagen cat.# 80224) according to the protocol provided by the supplier, with 30-mg tissue as input. The tissue was manually minced with a scalpel on ice followed by lysis and homogenization using TissueLyzer LT (Qiagen cat.# 85600) and Qiashredder (Qiagen cat.# 79654), respectively. Nucleic acid concentrations were measured by Nanodrop ND-1000 spectrophotometer, and RNA integrity was analyzed using Agilent 2100 Bioanalyzer (Agilent Technologies cat.# G2939BA).