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{"sheet":"Sheet1","index":0,"n_rows":105,"n_cols":3,"rows_kept":105,"flags":["formulas_cached_only"]}
["ITEM TO CHECK","IMPORTANCE","CHECKLIST"]
["EXPERIMENTAL DESIGN"]
["Definition of experimental and control  groups","E","Yes (see manuscript for details)"]
["Number within each group","E","Yes (see manuscript for details)"]
["Assay carried out by core lab or investigator's lab?","D","Yes (see manuscript for details)"]
["Acknowledgement of authors' contributions ","D","Yes (see manuscript for details)"]
["SAMPLE"]
["Description","E","Yes (see manuscript for details)"]
["     Volume/mass of sample processed","D","Yes (see manuscript for details)"]
["    Microdissection or macrodissection","E","Yes (see manuscript for details)"]
["Processing procedure","E","Yes"]
["     If frozen - how and how quickly?","E","Tumour biopts were snap frozen in liquid N2 immediately after resection and stored at -80 °C"]
["     If fixed - with what, how quickly?","E","Not applicable"]
["Sample storage conditions and duration (especially for FFPE samples)","E","All RNA samples were stored at -80°C"]
["NUCLEIC ACID EXTRACTION"]
["Procedure and/or instrumentation","E","Yes (see manuscript for details)"]
["     Name of kit and details of any modifications","E","Yes (see manuscript for details)"]
["     Source of additional reagents used ","D","Not applicable"]
["Details of DNase or RNAse treatment","E","DNAse treatment is not required as stem-loop miRNA reverse transcription only amplifies the mature miRNAs (see reference 10 in manuscript)"]
["Contamination assessment (DNA or RNA)","E","(see comment above)"]
["Nucleic acid quantification ","E","Yes (see manuscript for details)"]
["     Instrument and method","E","Yes (see manuscript for details)"]
["     Purity (A260/A280) ","D","No"]
["     Yield","D","No"]
["RNA integrity method/instrument","E","Yes (see manuscript for details)"]
["    RIN/RQI or Cq of 3' and 5' transcripts ","E","Yes (see manuscript for details)"]
["    Electrophoresis traces","D","No"]
[" Inhibition testing (Cq dilutions, spike or other) ","E","No (MIQE guidelines state that for screening studies these specifications are not required)"]
["REVERSE TRANSCRIPTION"]
["Complete reaction conditions","E","Yes (see manuscript for details)"]
["     Amount of RNA and reaction volume","E","Yes (see manuscript for details)"]
["    Priming oligonucleotide (if using GSP) and concentration","E","No (Manufacturer does not provide stem-loop RT primer sequences)"]
["     Reverse transcriptase and concentration","E","Yes (see manuscript for details)"]
["     Temperature and time","E","Yes (see manuscript for details)"]
["     Manufacturer of reagents and catalogue numbers","D","Yes (see manuscript for details)"]
["Cqs with and without RT","D*","No (see comment on DNAse treatment)"]
["Storage conditions of cDNA","D","cDNA was stored at -20°C "]
["qPCR TARGET INFORMATION"]
["If multiplex, efficiency and LOD of each assay.","E","No (MIQE guidelines state that for screening studies these specifications are not required)"]
["Sequence accession number","E","miRNA accession numbers are available in the mirbase registry"]
["Location of amplicon","D","No (Manufacturer does not provide this information)"]
["     Amplicon length","E","No (Manufacturer does not provide this information)"]
["     In silico specificity screen (BLAST, etc)","E","No (MIQE guidelines state that for screening studies these specifications are not required)"]
["     Pseudogenes, retropseudogenes or other homologs?","D","No (MIQE guidelines state that for screening studies these specifications are not required)"]
["          Sequence alignment","D","No (MIQE guidelines state that for screening studies these specifications are not required)"]
["     Secondary structure analysis of amplicon","D","No (MIQE guidelines state that for screening studies these specifications are not required)"]
["Location of each primer by exon or intron (if applicable)","E","No (Manufacturer does not provide this information)"]
["     What splice variants are targeted?","E","No (Manufacturer does not provide this information)"]
["qPCR OLIGONUCLEOTIDES"]
["Primer sequences","E","No (Manufacturer does not provide this information)"]
["RTPrimerDB Identification Number ","D","Not available"]
["Probe sequences","D**","No (Manufacturer does not provide this information)"]
["Location and identity of any modifications","E","5’ reporter dye 6-FAM; 3' MGB quencher"]
["Manufacturer of oligonucleotides","D","Applied Biosystems"]
["Purification method","D","No"]
["qPCR PROTOCOL"]
["Complete reaction conditions","E","Yes (see manuscript for details)"]
["     Reaction volume and amount of cDNA/DNA","E","Yes (see manuscript for details)"]
["     Primer, (probe), Mg++ and dNTP concentrations","E","No (Manufacturer does not provide this information)"]
["     Polymerase identity and concentration ","E","No (Manufacturer does not provide this information)"]
["     Buffer/kit identity and manufacturer ","E","Yes (see manuscript for details)"]
["     Exact chemical constitution of the buffer","D","No (Manufacturer does not provide this information)"]
["     Additives (SYBR Green I, DMSO, etc.)","E","Not applicable"]
["Manufacturer of plates/tubes and catalog number","D","Yes (see manuscript for details)"]
["Complete thermocycling parameters","E","Yes (see manuscript for details)"]
["Reaction setup (manual/robotic)","D","Yes (see manuscript for details)"]
["Manufacturer of qPCR instrument","E","Yes (see manuscript for details)"]
["qPCR VALIDATION"]
["Evidence of optimisation (from gradients) ","D","No (MIQE guidelines state that for screening studies these specifications are not required)"]
["Specificity (gel, sequence,  melt, or digest)","E","No (MIQE guidelines state that for screening studies these specifications are not required)"]
["For SYBR Green I, Cq of the NTC","E","Not applicable"]
["Standard curves with slope and y-intercept","E","No (MIQE guidelines state that for screening studies these specifications are not required)"]
["     PCR efficiency calculated from slope","E","No (MIQE guidelines state that for screening studies these specifications are not required)"]
["     Confidence interval for PCR efficiency or standard error","D","No (MIQE guidelines state that for screening studies these specifications are not required)"]
["     r2 of standard curve","E","No (MIQE guidelines state that for screening studies these specifications are not required)"]
["Linear dynamic range","E","Yes (see reference 9 and 10 in manuscript)"]
["     Cq variation at lower limit","E","No (MIQE guidelines state that for screening studies these specifications are not required)"]
["     Confidence intervals throughout range","D","No (MIQE guidelines state that for screening studies these specifications are not required)"]
["Evidence for limit of detection ","E","Yes (see reference 10 in manuscript)"]
["If multiplex, efficiency and LOD of each assay.","E","No (MIQE guidelines state that for screening studies these specifications are not required)"]
["DATA ANALYSIS"]
["qPCR analysis program (source, version)","E","Yes (see manuscript for details)"]
["     Cq method determination","E","Yes (see manuscript for details)"]
["     Outlier identification and disposition","E","No PCR replicates"]
["Results of NTCs ","E","No (NTCs were not included in the final screen; only in pilot study)"]
["Justification of number and choice of reference genes","E","Yes (see manuscript for details)"]
["Description of normalisation method","E","Yes (see manuscript for details)"]
["Number and concordance of biological replicates","D","Yes (see manuscript for details)"]
["Number and stage (RT or qPCR) of technical replicates","E","Yes (see manuscript for details)"]
["Repeatability (intra-assay variation)","E","No (MIQE guidelines state that for screening studies these specifications are not required)"]
["Reproducibility (inter-assay variation, %CV)","D","No (MIQE guidelines state that for screening studies these specifications are not required)"]
["Power analysis","D","No"]
["Statistical methods for result significance","E","Yes (see manuscript for details)"]
["Software (source, version)","E","Yes (see manuscript for details)"]
["Cq or raw data submission using RDML","D","Yes (see manuscript for details)"]
[]
["Table 1. MIQE checklist for authors, reviewers and editors. All essential information (E) must be submitted with the manuscript.  Desirable"]
["information (D) should be submitted if available. If using primers obtained from RTPrimerDB, information on qPCR target, oligonucleotides,"]
["protocols and validation is available from that source."]
[]
["*: Assessing the absence of DNA using a no RT assay is essential when first extracting RNA. Once the sample has been validated as"]
[" RDNA-free, inclusion of a no-RT control is desirable, but no longer essential."]
[]
["**: Disclosure of the probe sequence is highly desirable and strongly encouraged. However, since not all commercial pre-designed assay"]
[" vendors provide this information, it cannot be an essential requirement. Use of such assays is advised against."]