RNA was extracted from each of the clinical research samples using the AllPrep DNA/RNA FFPE Kit (Qiagen, Valencia, CA, USA) based on the respective standard extraction procedures. RNA was quantified using the Nanodrop 2000 instrument (Thermo Fisher Scientific, Wilmington, DE, USA) and the RiboGreen RNA Assay Kit (Thermo Fisher Scientific).
Allprep dna rna ffpe kit
Manufactured by Qiagen
Sourced in Germany, United States, Netherlands, Sweden, United Kingdom, Canada, Spain
The AllPrep DNA/RNA FFPE Kit is a laboratory product designed to extract and purify both DNA and RNA from formalin-fixed, paraffin-embedded (FFPE) samples. The kit provides a standardized protocol for simultaneous isolation of genomic DNA and total RNA from the same FFPE tissue sample.
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Lab products found in correlation
Qubit dsdna hs assay kit, by Thermo Fisher Scientific (23 mentions)
Nanodrop 2000, by Thermo Fisher Scientific (20 mentions)
Nanodrop, by Thermo Fisher Scientific (19 mentions)
Allprep dna rna mini kit, by Qiagen (16 mentions)
Qubit fluorometer, by Thermo Fisher Scientific (15 mentions)
Allprep dna rna mirna universal kit, by Qiagen (14 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (14 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (11 mentions)
Qubit 3.0 fluorometer, by Thermo Fisher Scientific (11 mentions)
Nanodrop 1000, by Thermo Fisher Scientific (10 mentions)
Nanodrop 2000 device, by Thermo Fisher Scientific (8 mentions)
Qubit 2.0 fluorometer, by Thermo Fisher Scientific (8 mentions)
Nanodrop nd 1000 spectrophotometer, by Thermo Fisher Scientific (8 mentions)
Agilent 2200 tapestation system, by Agilent Technologies (7 mentions)
2100 bioanalyzer, by Agilent Technologies (7 mentions)
Qubit rna hs assay kit, by Thermo Fisher Scientific (7 mentions)
Deparaffinization solution, by Qiagen (7 mentions)
Nanodrop 1000 spectrophotometer, by Thermo Fisher Scientific (7 mentions)
Allprep dna rna kit, by Qiagen (6 mentions)
Sensifast sybr lo rox kit, by Meridian Bioscience (6 mentions)
387 protocols using allprep dna rna ffpe kit
1
RNA Extraction from FFPE Samples
2
FFPE RNA Extraction and Quantitative PCR
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DNA and total RNA were extracted from formalin-fixed paraffin-embedded (FFPE) sections with the AllPrep® DNA/RNA FFPE Kit (Qiagen) according to manufacturer’s instructions. Template RNA was used to prepare cDNA with random primers (Ambion) and Sensiscript Reverse Transcriptase (Qiagen). Real time PCR was carried out in 96-well plates using the 7900® ABI Sequence Detection System (Applied Biosystems) and the QuantiTectTM SYBR® Green PCR Kit (Qiagen) according to the manufacturer’s protocols. Primers previously identified for assessment of hESC differentiation towards renal lineages were utilized [24 (link)] as markers for pluripotency (octamer binding protein 4, OCT4), posterior primitive streak (brachyury, BRY), intermediate mesoderm (odd skipped-related transcription factor one, OSR1; LIM homeobox protein 1, LIM1; PAX2), and metanephric mesenchyme (SIX2, PAX2, WT1).
3
Laser Capture Microdissection and RNA Sequencing of Mouse Sebaceous Glands
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Mouse back skin from CR, GF, CR×CR F1, and GF×GF F1 was collected and fixed overnight in 4% paraformaldehyde (Fisher AAJ19943K2) at 4°C followed by paraffin embedding. Laser capture microdissection (LCM) was performed using the LMD 7000 system (Leica Microsystems). FFPE mouse skin was processed and cut onto a polyethylene naphthalate (PEN) slide designed for LCM processing (Leica 11505158). At least 1,000 SGs or 1,000,000 μm2 of tissue was isolated to obtain enough material for RNA extraction. SG RNA was extracted from post-LCM tissue using a Qiagen All Prep DNA/RNA FFPE Kit (Qiagen 80234). RNA concentration was measured by Qubit fluorometric quantification (ThermoFisher Qubit 2.0 Fluorometer) and RNA quality measured via BioAnalyzer (Agilent 2100 Bioanalyzer Instrument). cDNA libraries were prepared using Illumina Stranded Total RNA Prep with Ribo-Zero Plus Kit (Illumina 20040529) with IDT for Illumina RNA UD Indexes, Set A (Illumina 20040553). Libraries were assessed for cDNA quantity and library quality using Qubit and BioAnalyzer. As necessary, an extra bead wash step was performed to remove excess primer dimers in the library and purify samples further. Samples were then pooled and sequenced on a Nextseq 550 using a NextSeq 500/550 High Output Kit v2.5 (150 Cycles) (Illumina 20024907).
4
FFPE DNA/RNA Extraction and Mutational Analysis
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The extraction and purification of DNA and RNA from FFPE samples were performed using the Qiagen AllPrep DNA/RNA FFPE kit according to the manufacturer’s instructions. Briefly, fresh FFPE tissue (2–4 sections of 10–20 µm) containing > 50% tumor cells were deparaffinized and incubated in a lysis buffer containing proteinase K. The mixture was centrifuged to precipitate the DNA, leaving the RNA in the supernatant. In addition, freshly cut FFPE tissue (10–20 µm sections) containing normal cells was used for similar DNA and RNA extraction. DNA quality control was carried out using a 260 nm/280 nm ratio assessment and 2% agarose gel electrophoresis. RNA samples were preserved for future studies. The mutational profile of tumor DNA was analyzed using the EntroGen® Colon Cancer mutation detection panel (CRC-RT48), specifically designed for tumor DNA. Genotyping of drug-metabolism-related genes was conducted using the TaqMan® assay with germline DNA. The selected polymorphisms for this study were chosen based on their relevance in previous research. To validate germline polymorphism results, assays were performed on both tumor DNA and germline DNA, revealing consistent findings. Further details on the TaqMan® assays can be found in the supplementary material .
5
RNA Extraction and RNA-seq Library Prep
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RNA extraction was carried out with AllPrep DNA/RNA FFPE Kit (Qiagen, #80234) with modifications described previously17 (link). RNA-seq libraries were prepared with Trio RNA-Seq library preparation kit (Tecan, #0506-A01), quantified with Qubit dsDNA HS assay kit (ThermoFisher #Q32851 ) and pooled for sequencing on NovaSeq6000 (SP 2 x 150 bp).
6
FFPE DNA Extraction and Quantification
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Genomic DNA was extracted from FFPE tissue sections (generally 6–10 mm in size) and purified using a Qiagen AllPrep DNA/RNA FFPE Kit (Qiagen, Venlo, The Netherlands). Qubit dsDNA HS Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA) was used for quantitating DNA concentration, and 120 ng of input DNA was used for library preparation after modification of the manufacturer’s instructions. We used Genomic DNA ScreenTape (Agilent Technologies, Santa Clara, CA, USA) on an Agilent 2200 TapeStation system (Agilent Technologies, Santa Clara, CA, USA) to assess integrity, fragment size, and quality of DNA.
7
Amplicon Sequencing of FFPE Samples
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DNA were purified with the use of an Allprep DNA/RNA FFPE Kit (Qiagen, Valencia, CA, USA) according to the manufacturer’s instructions. Amplicon sequencing were performed using the Ion AmpliSeq Colon and Lung Cancer Panel (Thermo Fisher Scientific, Wilmington, DE, USA). Detailed procedure was described in Supplementary Method.
8
Microsatellite Instability Analysis in Tumor Tissues
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DNA was isolated from snap-frozen tumor tissues using an All-prep DNA/RNA mini kit (Cat # 80204 Qiagen, Sollentuna, Sweden), or from FFPE tumor tissue using an All-prep DNA/RNA FFPE kit (Cat # 80234; Qiagen, Sollentuna, Sweden). The MSI status was analyzed using the MSI Analysis System, version 1.2 (Promega, Madison, USA), which examined five microsatellites. The PCR was run on the Perkin-Elmer Gene Amp PCR system 9600 Thermal Cycler (Perkin Elmer, USA) according to the manufacturer’s instructions using 2 ng of DNA. The MSI markers were detected on an ABI prism 3730 instrument at KI Gene using the PowerPlex 4C matrix Standard (Cat # DG4800; Applied Biosystems, USA). MSI was defined as peak alterations in the marker electropherogram when tumor tissue was compared with matching mucosa. When more than one marker showed instability, the tumor was defined as MSI-H. If only one marker showed instability, it was defined as MSI-L. If no instability was detected, the tumor was designated as MSS.
9
PathoChip Screening for Cancer Diagnostics
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The PathoChip screening procedure has been previously described [10 ,11 ,[16] (link), [17] (link), [18] (link)]. DNA and RNA were simultaneously extracted from cancer patients and control FFPE tissue samples using the AllPrep DNA/RNA FFPE Kit (Qiagen, Hilden, Germany), and absorbance was measured at 260/280 nm to assess quality. Patient sample RNA (100 ng) and DNA (50 ng) were used for whole genome and transcriptome amplification (WTA) using the TransPlex Complete Whole Transcriptome Amplification Kit (Sigma-Aldrich, St. Louis, MO). Human DNA and RNA were extracted from the human B cell line BJAB (obtained from ATCC) to serve as a reference for cross-hybridization of probes to the amplified patient genomes and to determine background normalization. Purified WTA product (PCR purification kit, Qiagen, Germantown, MD, USA) quality was determined using A260/280 measurements, and 1 μg was labeled with either Cy3 (amplified patient sample) or Cy5 (amplified human reference) (SureTag labeling kit, Agilent Technologies, Santa Clara, CA). All labeled specimens were purified, and a Cy3-labeled and Cy5-labeled reference were hybridized together on each PathoChIP array in constant rotation at 65 °C for 40 h, as previously described [10 ,11 ]. Array slides were washed and then scanned for visualization using an Agilent SureScan G4900DA array scanner.
10
Tumor Tissue DNA and Plasma ctDNA Isolation
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Archived formalin-fixed, paraffin-embedded (FFPE) tissue specimens obtained at the time of diagnosis were collected. Blood samples (14 ml) were collected into tubes containing EDTA just before the first and second cycle and at the end of the protocol treatment; the samples were then centrifuged at 1200 × g for 10 min within 1 h after collection. Tumor tissue DNA in FFPE was isolated using the Allprep DNA/RNA FFPE Kit (Qiagen, Valencia, CA) according to the manufacturer’s instructions. Plasma ctDNA was isolated using the cobas® cfDNA Sample Preparation Kit (Roche Diagnostics Ltd., Penzberg, Germany) according to the manufacturer’s instructions. The quality and quantity of the nucleic acid were verified using PicoGreen dsDNA Reagent (all from Thermo Scientific, Wilmington, DE).
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