In order to ensure experimental transparency, consistency between research laboratories and thus, maintain a high level of integrity in publications, a guideline for real-time PCR experiments has been edited in 2009 by an international research team [PMID: 19246619].
Considering the increasing number of publications in the Digital PCR field, the MIQE (for Minimum Information for Publication of Quantitative real-time PCR Experiments) guideline has been updated for digital PCR experiments under the name “the Minimum Information for the Publications of Quantitative Digital PCR Experiments guidelines (dMIQE)”[PMID: 23570709].
Having a look at this guideline before starting designing an experiment can be very informative. Indeed, as an example of what you can read in the dMIQE, you will find, below (Table 1), a checklist you might follow for performing a digital PCR experiment. Finally, all items are categorized as essential (E) or desirable (D) for manuscript submission. Thus, citing the following dMIQE in your publication will be a guarantee of quality, for reviewers and readers.
|Table 1. dMIQE checklist for authors, reviewers and editors.a|
|Item to check||Importance||Item to check 2||Importance|
|Experimental design||dPCR oligonucleotides|
|Definition of experimental and control groups.||E||Primer sequences and/or amplicon context sequence.b||E|
|Number within each group.||E||RTPrimerDB (real-time PCR primer and probe database) identification number.||D|
|Assay carried out by core lab or investigator’s lab ?||D||Probe sequences.b||D|
|Power analysis.||D||Location and identify of any modifications.||E|
|Sample||Manufacturer of oligonucleotides.||D|
|Volume or mass of sample processed.||E||dPCR protocol|
|Microdissection or microdissection.||E||Complete reaction conditions.||E|
|Processing procedure.||E||Reaction volume and amount of RNA/cDNA/DNA||E|
|If frozen-how and how quickly?||E||Primer, (probe), Mg ++ and dNTP concentrations.||E|
|If fixed- with what, how quickly?||E||Polymerase identity and concentration.||E|
|Sample storage conditions and duration (especially for formalin-fixed, paraffin-embedded samples).||E||Buffer/kit catalogue no. and manufacturer.||E|
|Nucleic acid extraction||Exact chemical constitution of the buffer||D|
|Quantification-instrument/method.||E||Additives (SYBR green I, DMSO, etc.).||E|
|Storage conditions: temperature, concentration, duration, buffer.||E||Plates/tubes Catalogue No and manufacturer.||D|
|DNA or RNA quantification||E||Complete thermocycling parameters.||E|
|Quality/integrity, instrument/method, e.g. RNA integrity/R quality index and trace or 3’:5’.||E||Reaction setup.||D|
|Template structural information.||E||Gravimetric or volumetric dilutions (manual/robotic).||D|
|Template modification (digestion, sonication, preamplification, etc.)||E||Total PCR reaction volume prepared.||D|
|Template treatment (initial heating or chemical denaturation).||E||Partition number.||E|
|Inhibition dilution or spike.||E||Individual partition volume.||E|
|DNA contamination assessment of RNA sample.||E||Total volume of the partitions measured (effective reaction size).||E|
|Detail of DNase treatment where performed.||E||Partition volume variance/SD.||D|
|Manufacturer of reagents used and catalogue number||D||Comprehensive details and appropriate use of controls.||E|
|Storage of nucleic acid: temperature, concentration, duration, buffer.||E||Manufacturer of dPCR instrument.||E|
|RT (if necessary)||dPCR validation|
|cDNA priming method + concentration.||E||Optimization data for the assay.||D|
|One- or 2-step protocol.||E||Specificity (when measuring rare mutations, pathogen sequences etc.)||E|
|Amount of RNA used per reaction||E||Limit of detection of calibration control.||D|
|Detailed reaction components and conditions.||E||If multiplexing, comparison with singleplex assays.||E|
|RT efficiency.||D||Data analysis|
|Estimated copied measured with and without addition of RT.b||D||Mean copies per partition (ʎ or equivalent).||E|
|Manufacturer of reagents used and catalogue number.||D||dPCR analysis program (source, version).||E|
|Reaction volume (for 2-step RT reaction).||D||Outlier identification and disposition.||E|
|Storage of cDNA : temperature, concentration, duration, buffer.||D||Results of no-template controls.||E|
|dPCR target information||Examples of positive(s) and negative experimental results as supplemental data.||E|
|Sequence accession number.||E||Where appropriate, justification of number and choice of reference genes.||E|
|Amplicon length.||E||Number and concordance of biological replicates.||D|
|In silico specificity screen (BLAST, etc.)||E||Number and stage (RT or dPCR) of technical replicates.||E|
|Pseudogenes, retropseudogenes or other homologs?||D||Repeatability (intraassay variation).||D|
|Sequence alignment.||D||Reproductibility (interassay/user/lab etc. variation).||D|
|Secondary structure analysis of amplicon and GC content.||D||Experimental variance or Cl.d||E|
|Location of each primer by exon or intron (if applicable).||E||Statistical methods used for analysis.||E|
|Where appropriate, which splice variants are targeted?||E||Data submission using RDML (Real-time PCR Data Markup Language).
|a All essential information (E) must be submitted with the manuscript. Desirable information (D) should be submitted if possible.
b Disclosure of the primer and probe sequence is highly desirable and strongly encouraged. However, since not all commercial predesigned assay vendors provide this information, when it is not available assay context sequences must be submitted [Bustin et al. (48)].
c Assessing the absence of DNA using a no-RT assay (or where RT has been inactivated) is essential when first extracting RNA. Once the sample has been validated as DNA free, inclusion of a no-RT control is desirable, but no longer essential.
d When single dPCR experiments are performed, the variation due to counting error alone should be calculated from the binomial (or suitable equivalent) distribution.