Samples as well as the positive control were measured in triplicates whereas non-template controls (NTC) and non reverse transcriptase controls (NRTC) were measured in duplicates. Melt curve analysis included a 30 sec hold at 65 ☌ followed by a gradual increase to 95 ☌ with a temperature increment of 0.5☌/ 5 sec. Each amplification cycle implied a denaturation step at 95 ☌ for 10 sec and a primer annealing/ elongation step at 60 ☌ for 30 sec. The thermal cycling protocol included a single polymerase activation step at 95 ☌ for 2 min followed by 40 amplification cycles as well as melt curve analysis. SYBR® Green supermix (Biorad, Hilden) + 1 µl 20x PrimePCR™ SYBR® Green Assay (Primer, Biorad, Hilden). QPCR Protocol Complete reaction conditions
Reaction: 2 µl cDNA (1/10 dilution of original cDNA) + 7 µl RNase free water + 10 µl 2x SsoAdvanced universal Location and identity of any modifications S3 Table, Biorad provided Amplicon Context Sequence Not applicable, but Biorad Unique Assay ID is listed in S3 Table QPCR Oligonucleotides Primer sequences RTPrimerDB Identification Number Probe sequences Not applicable but Biorad key requirements for primer design were published in "PrimePCR_Assay_Validation_Tech_Note_6262" "PrimePCR_Assay_Validation_Tech_Note_6262" Location of each primer by exon or intron (if applicable) What splice variants are targeted? Yes, but not applicable but Biorad key requirements for primer design were published in "PrimePCR_Assay_Validation_Tech_Note_6262" "PrimePCR_Assay_Validation_Tech_Note_6262" not applicable but Biorad key requirements for primer design were published in "PrimePCR_Assay_Validation_Tech_Note_6262" Sequence alignment Pseudogenes, retropseudogenes or other homologs? Yes, but not applicable but Biorad key requirements for primer design were published in In silico specificity screen (BLAST, etc) If multiplex, efficiency and LOD of each assay. QuantiTec Reverse Transcription Kit (Cat.No: 205311, Qiagen, Hilden) Specified in "Complete reaction conditions" Also contains RNase inhibitor (Qiagen, Hilden) Temperature and time Manufacturer of reagents and catalogue numbers Hilden) 1 µl of Quantiscript® Reverse Transcriptase: A mixture of the QIAGEN® products Omniscript® Reverse Transcriptase and Sensiscript® Reverse Transcriptase. Priming oligonucleotide (if using GSP) and concentrationġ µl RT Primer Mix (Qiagen, Hilden): optimized blend of oligo-dT and random primers dissolved in water (Qiagen, Reverse Transcription Complete reaction conditionsġ2 µl Rnase-free water (Qiagen) incubation 42☌ for 2 min, 2.step: samples were mixed with 1µl Quantiscript Reverse Transcriptase+4 µl 5xQuantiscript RT Buffer+1 µl RT Primer Mix incubation 42☌ for 15 min followed by 95☌ for 3 min on iCycler Thermal Cycler (Biorad, Munich) Amount of RNA and reaction volume Inhibition testing (Cq dilutions, spike or other)ġ.step: 2 µl 7x gDNA Wipeout Buffer (for effective elimination of genomic DNA contamination)+1 µg total RNA in NanoDrop 2000 (Thermo Scientific, USA), UV-Vis spectrophotometerĭNase I stock solution, incubation 10min room temperature (~2.7 Kunitz per 100 µl)īioanalyzer 2100 (Agilent Technologies, USA) Non reverse transcriptase controls (NRTC) were performed in qPCR and no Cq values or Cq ≥ 37 were detected In solution digestion of genomic DNA 87.5µl RNA solution (RNA content ≤ 45 µg) + 10 µl Buffer RDD + 2.5 μl QIAzol® Lysis reagent, RNase-Free DNase Set, RNeasy MinElute Cleanup Kit (Qiagen, Hilden)Ĭhloroform, Isopropanol (Sigma, Steinheim)
Sample storage conditions and duration (especially for FFPE samples) Nucleic Acid Extraction Procedure and/or instrumentationĪccording to the supplier’s instructions Name of kit and details of any modifications phenol-chloroform extraction (1ml QIAzol/ sample), 2. Resected tissue samples were immediatly frozen in liquid nitrogen at the 4 ischemia time pointsįrozen in liquid nitrogen until RNA preparationġ. Resected tissue samples were frozen in liquid nitrogen at the indicated time points. Each tissue sample had a weightīetween 120 - 200 mg or a size of at least 5 x 5 x 5 mm (=125 mm3 = 0.125 cm3).
Normal tissue and tumor tissue were collected at each specified time point.
Samples from normal tissue and tumor tissue were collected at each specified time point.ġ20 - 200 mg or a size of at least 5 x 5 x 5 mm Tissue samples (colorectal) were collected from 20 patients and processed. MIQE checklist for authors, reviewers and editors.Ģ0 cases for 4 ischemia time points from normal and corresponding tumor colon tissueĪssay carried out by core lab or investigator's lab?Īcknowledgement of authors' contributionsĭefinition of experimental and control groups