The H and E stained slide was used for histopathological review and to guide tumor macrodissection of sections from four unstained slides. The unstained slides from samples with ≥ 40% tumor cellularity were incubated at 65 °C for 30 minutes and deparaffinized using Citrisolv (Fisher Scientific, Pittsburgh, PA) followed by ethanol wash. Tumor tissues were macrodissected from the slides into RNAse-free microfuge tubes, and nucleic acids isolated using the Qiagen AllPrep FFPE kit (#80234). Manufacturer’s instructions were followed with the exception that the proteinase K digestion step was extended to an overnight incubation for the DNA isolation. Total RNA and DNA were quantified using the Invitrogen Qubit and corresponding quantification kits. Total RNA was used for the miRGE assay (see below). DNA pellets were stored at −80° C for future use.
Allprep ffpe kit
Manufactured by Qiagen
Sourced in Germany, United States
The AllPrep FFPE kit is a laboratory product designed to extract and purify total RNA, DNA, and miRNA from formalin-fixed, paraffin-embedded (FFPE) tissue samples. The kit utilizes a column-based technology to efficiently isolate the desired biomolecules from FFPE samples.
Automatically generated - may contain errors
Lab products found in correlation
Corresponding quantification kits, by Thermo Fisher Scientific (2 mentions)
Citrisolv, by Thermo Fisher Scientific (2 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (2 mentions)
Rneasy mini kit, by Qiagen (2 mentions)
Trusight oncology 500, by Illumina (2 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (2 mentions)
Nebnext small rna kit, by New England Biolabs (1 mentions)
Hiseq 2500 platform, by Illumina (1 mentions)
Rna 6000 pico, by Agilent Technologies (1 mentions)
Quant it ribogreen assay, by Thermo Fisher Scientific (1 mentions)
Quant it dsdna broad range assay kit, by Thermo Fisher Scientific (1 mentions)
Eb buffer, by Qiagen (1 mentions)
Agilent bioanalyzer rna nanochip, by Agilent Technologies (1 mentions)
Allprep dna rna ffpe kit, by Qiagen (1 mentions)
Rt2 rna qc pcr array, by Qiagen (1 mentions)
Rneasy ffpe kit, by Qiagen (1 mentions)
Qubit dsdna high sensitivity kit, by Thermo Fisher Scientific (1 mentions)
Rna 6000 pico kit, by Agilent Technologies (1 mentions)
Sensifast probe lo rox kit, by Meridian Bioscience (1 mentions)
Taqman microrna reverse transcription kit, by Thermo Fisher Scientific (1 mentions)
21 protocols using allprep ffpe kit
1
Macrodissection and Nucleic Acid Isolation from FFPE Tumor Tissue
2
Gallbladder Tissue RNA-seq Protocol
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Formalin-fixed, paraffin-embedded (FFPE) gallbladder tissue specimens were obtained from 98 patients in total (n = 31 GS; n = 35 Dys; n = 32 GBC). RNA was extracted from FFPE sections using the AllPrep FFPE kit following Qiagen’s recommendations, and RNA quality was controlled (High Sensitivity Genomic DNA, Advanced Analytical, United States, and FFPE quality control kits, Illumina).
The NEBNext Small RNA kit (NEB) was used to produce RNA sequencing libraries, which were sequenced on the HiSeq 2500 platform (Illumina, San Diego, CA, USA) to an average depth of 18 M reads per sample. The applied RNA sequencing protocol has been previously described in detail [18 (link)]. Briefly, our protocol enabled us to capture lncRNA mapped fragments in the size range up to 47 base pairs. First, reads from the HiSeq 2500 platform were adapter-trimmed (AdapterRemoval v2.1.7) [19 (link)]. Then, adapter-trimmed reads were mapped to the human genome (hg38) by a Bowtie2 v2.2.9 aligner [20 (link)]. HTSeq was used to count reads mapped to lncRNA regions in GENCODE v26 annotations [21 (link),22 (link)].
The NEBNext Small RNA kit (NEB) was used to produce RNA sequencing libraries, which were sequenced on the HiSeq 2500 platform (Illumina, San Diego, CA, USA) to an average depth of 18 M reads per sample. The applied RNA sequencing protocol has been previously described in detail [18 (link)]. Briefly, our protocol enabled us to capture lncRNA mapped fragments in the size range up to 47 base pairs. First, reads from the HiSeq 2500 platform were adapter-trimmed (AdapterRemoval v2.1.7) [19 (link)]. Then, adapter-trimmed reads were mapped to the human genome (hg38) by a Bowtie2 v2.2.9 aligner [20 (link)]. HTSeq was used to count reads mapped to lncRNA regions in GENCODE v26 annotations [21 (link),22 (link)].
3
RNA Extraction and Sequencing from FFPE Samples
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The AllPrep FFPE Kit (Qiagen, Germantown, Maryland, USA) was utilized for the RNA extraction from archival FFPE samples for RNA sequencing in this study. RNA isolates were eluted in a 14 μl volume of RNase/DNase-free H2O. RNA was quantified utilizing the Quant-iT RiboGreen Assay (Life Technologies—cat# R11490).
RNA quality was assessed on Agilent Bioanalyer using RNA 6000 Pico or nano kit. Since RNA from FFPE are all degraded, DV200 analysis were performed instead of RIN (RNA integrity number) for a more accurate read out of RNA quality. DV200 is an analysis recommended by the RNA seq kit protocol, which calculates the percentage of RNA fragments larger than 200 nucleotides. A higher DV200 score indicates better RNA quality. For library preparation, varies quantity of RNA were used to compensate for quality variations following the Library construction kit (Illumina RNA Exome kit) instructions. For RNA with DV200 higher than 70%, 20 ng RNA was used for library construction; for DV200 between 50–70%, 40 ng; and for DV200 under 50%, 100 ng29 . All RNA library passed quality assessment before putting into whole transcriptome hybridization and capture. Post-capture libraries passed QC on TapeStation before loading into sequencer. Samples that disqualify were dismissed.
RNA quality was assessed on Agilent Bioanalyer using RNA 6000 Pico or nano kit. Since RNA from FFPE are all degraded, DV200 analysis were performed instead of RIN (RNA integrity number) for a more accurate read out of RNA quality. DV200 is an analysis recommended by the RNA seq kit protocol, which calculates the percentage of RNA fragments larger than 200 nucleotides. A higher DV200 score indicates better RNA quality. For library preparation, varies quantity of RNA were used to compensate for quality variations following the Library construction kit (Illumina RNA Exome kit) instructions. For RNA with DV200 higher than 70%, 20 ng RNA was used for library construction; for DV200 between 50–70%, 40 ng; and for DV200 under 50%, 100 ng29 . All RNA library passed quality assessment before putting into whole transcriptome hybridization and capture. Post-capture libraries passed QC on TapeStation before loading into sequencer. Samples that disqualify were dismissed.
4
DNA Extraction and Library Prep from FFPE
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DNA was extracted from each tumour core using the Qiagen AllPrep® FFPE kit. DNA samples were quantified using the Quant-iT™ broad range ds-DNA assay kit (Invitrogen™, Life Technologies, USA) according to manufacturer’s recommendations.
Whole-genome DNA libraries were prepared using either a previously described library preparation method9 (link),27 (link); or using the NEBNext® UltraTM DNA Library Prep kit for Illumina® (indexed primers) (New England BioLabs, UK). Automated library preparation using FFPE derived DNA was found to produce libraries of variable quality (data not shown) so all libraries were subsequently prepared manually. We used 5 DNA samples to ascertain that libraries of higher yield were prepared after bovine serum albumin (BSA) addition compared to libraries prepared from a second tumour core from the same samples without BSA addition (data not shown). Library preparation protocols were modified by adding 5 mg/ml BSA as a blocking agent to each reaction to reduce PCR inhibition previously ascribed to melanin8 (link).
Whole-genome DNA libraries were prepared using either a previously described library preparation method9 (link),27 (link); or using the NEBNext® UltraTM DNA Library Prep kit for Illumina® (indexed primers) (New England BioLabs, UK). Automated library preparation using FFPE derived DNA was found to produce libraries of variable quality (data not shown) so all libraries were subsequently prepared manually. We used 5 DNA samples to ascertain that libraries of higher yield were prepared after bovine serum albumin (BSA) addition compared to libraries prepared from a second tumour core from the same samples without BSA addition (data not shown). Library preparation protocols were modified by adding 5 mg/ml BSA as a blocking agent to each reaction to reduce PCR inhibition previously ascribed to melanin8 (link).
5
Precise FFPE RNA and DNA Extraction
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The FFPE tissues were deparaffinized using xylene and ethanol according to the procedures detailed by Qiagen for the AllPrep DNA/RNA FFPE Kit. RNA was eluted in 60 microliters EB buffer (Qiagen). Qiagen All Prep FFPE kit was used to purify DNA and RNA from each specimen; the entire tissue section sent was used for nucleic acid purification (i.e. no macrodissection was performed). RNA quality was checked by running on the Agilent Bioanalyzer (RNA Nano chip) and RNA Integrity Number (RIN) values were obtained. The RNA quality was also assessed using the RT2 RNA QC PCR Array from Qiagen that tests for the presence of transcripts of two housekeeping genes, ACTB and HPRT1, in addition to testing for inhibitors of Reverse Transcription and PCR amplification reactions, and the presence of genomic DNA contamination.
6
Nucleic Acid Isolation from Murine Tumors and Human PDAC Tissue
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Murine tumors DNA and RNA were isolated from fresh subcutaneous tumors according to previously published protocols from our institution [44 (link)].
Human PDAC tissue A board-certified pathologist reviewed the surgical pathology specimens and determined that the biospecimens from all three subjects had sufficient tumor cellularity to proceed with nucleic acid isolation. Five to six 1.5 mm punches were taken from formalin-fixed paraffin-embedded (FFPE) blocks from areas of estimated tumor cellularity of > 40%, using an H&E-stained slide as a guide. For nucleic acid isolation, RNA and DNA were purified from the punches using the Qiagen AllPrep FFPE Kit (catalog # 80,234, Germantown, MD, USA) following manufacturer’s instructions except for increased Proteinase K digestion length. As RNA degradation was a specific concern, we analyzed RNA integrity numbers (RIN) and fragment distribution values, DV200, which reflects the percentage of RNA fragments > 200 base pairs.
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7
Nucleic Acid Extraction from FFPE Tissue
8
FFPE Nucleic Acid Extraction Protocol
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Serial sections were evaluated for tumor proportion and necrosis prior to nucleic acid extraction. DNA and RNA were extracted using the QIAGEN (Valencia, CA) AllPrep FFPE kit. Genomic DNA was quantitated using the Qubit dsDNA high sensitivity (HS) kit (ThermoFisher Scientific, Waltham, MA); total RNA was quantitated and quality checked using the Agilent 2100 Bioanalyzer with the RNA 6000 pico kit (Agilent Technologies, Santa Clara, CA).
9
Tumor Macrodissection and Nucleic Acid Extraction
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Details on our assay were previously published [29 (link)]; for the sake of completeness, we briefly summarize the approach here. Archival tumor blocks were prepared into histological sections. (5 µm each). One slide was stained with hematoxylin and eosin for histopathological review and to guide tumor macrodissection from four unstained sections. The remaining unstained slides from samples with ≥ 40% tumor cellularity were incubated at 65 °C for 30 min and deparaffinized using Citrisolv (Fisher Scientific, Pittsburgh, PA) followed by ethanol wash. Tumor tissues were macrodissected into RNAse-free microfuge tubes, and nucleic acids isolated using the Qiagen AllPrep FFPE kit (#80234). Manufacturer’s instructions were followed with the exception that the proteinase K digestion step was extended to an overnight incubation for DNA isolation. Total RNA and DNA were quantified using the Invitrogen Qubit and corresponding quantification kits. DNA pellets were stored at − 80° C for future use.
10
Quantitative miRNA Expression Analysis
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