106:
confirmation of effectiveness for said data. These studies retained major reproducibility issues, where the conclusions of their evidence could not be replicated by other researchers, throwing the initial results into doubt. All of this was despite many papers directly citing Bustin's original MIQE publication, but not following through on the guideline checklist of material in their own experiments. However, some researchers have pointed out at least some success, with a number of papers being rejected by academic journals for publication due to failing to pass MIQE checklists. Other studies have been retracted after the fact once their lack of proper data to pass the MIQE guidelines was noted and publicly pointed out to the journal editors.
57:
suggestions on basic experimental procedures and forms of data that should be collected as a minimum level of reported information for other researchers to understand and use when reading the published material. Setting up a recognized and largely agreed upon set of guidelines such as these were deemed important by the scientific community especially due to the ever increasing amount of scientific work coming from
161:
experiment. These two pieces are defined as essential for any study. This section also includes two desirable points, which are pointing out whether the author's laboratory itself or a core laboratory of the university or organization conducted the qPCR assay and an acknowledgement of any other individuals that contributed to the work.
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to put together a set of guidelines on how to perform qPCR and what forms of data should be collected and published in the process. This also allowed editors and reviewers of scientific journals to employ the guidelines when looking over a submitted paper that included qPCR data. Thus, the guidelines
169:
The essential requirements that samples and sample material must meet includes a description of the sample, what form of dissection was used, what processing method was done, whether the samples were frozen or fixed and how long did it take, and what sample conditions were used. It is also desirable
130:
An overview of the 10th anniversary since the publication of the MIQE guidelines was conducted in June 2020 and discussed the scientific studies that had produced better and more organized results when following the guidelines. In August 2020, an updated version of the guidelines for the digital PCR
312:
In order to confirm the effectiveness and quality of the qPCR process that was performed, there are several actions and subsequent data that must be presented. This includes explaining the specific method of checking that the process functioned, such as using a gel, direct sequencing of the genetic
277:
Creation of the oligonucleotides requires only two pieces of essential information: the primer sequences used and the location and details of any modifications made to the sequence. But there are several desirable pieces of data, including the identification number from the RTPrimerDB database, the
187:
treatment was used, a statement on whether any contamination was assessed, a quantification of the amount of genetic material extracted, a description of the instruments used for the extraction, the methods used to retain RNA integrity, a statement on the RNA integrity number and quality indicator
349:
The final section of the guidelines involves information on how the analysis of the qPCR data was done. The essential parts of that include the program and program version used for the analysis, the method for how the Cq was determined, figuring out the outlier points in the data and how they are
325:
was used, then the Cq of the control group with no template DNA must be given. Further essential data includes the calibration of the machine curves with the slope and y intercept noted, the efficiency of the PCR process as determined from the aforementioned slope, the correlation coefficients (r
160:
Large portions of the guidelines include basic actions that would normally be included in experiments and publications regardless, such as an item for describing the experimental and control group differences. Other such information includes how many individual units are used in each group in the
105:
It was noted by Bustin in 2014 (and again by him in 2017) that there was some amount of uptake and usage of the MIQE guidelines within the scientific community, but there were still far too many published papers with qPCR experiments that lacked even the most basic of data presentation and proper
118:
stated that they had designed the data collection portion around the MIQE guidelines so that the data fit all the minimum parameter checklists in the protocols. Other scientific instrument companies have assisted in guideline compliance by purposefully tailoring their devices for them, including
56:
machinery allowed for such experiments to be run for a cheaper cost. Because the technique is utilized across all of science in multiple fields, the instruments, methods, and designs of how qPCR is used differ greatly. To help improve overall quality, the MIQE guidelines were made as generalized
151:
The MIQE guidelines are split up into 9 different sections that make up the checklist. These include not only considerations for doing the qPCR itself, but also how the resulting data is collected, analyzed, and presented. An important part of the latter is including information relating to the
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As one of the primary segments of the guidelines, there are several essential parts on the checklist for the qPCR process itself. This includes the full set of conditions used for the reaction, the volume of both the reaction and the cDNA, the concentrations for the probes, magnesium ions, and
39:
through the use of RT-qPCR, but the results proved to be completely unreproducible by other scientists. The authors themselves also did not try to reproduce them and the raw data was found to have a large amount of errors and basic mistakes in analysis. This incident prompted
131:
method was published to account for improvement in machinery, technologies, and techniques since the original 2013 release. Additional guideline steps were added for data analysis, while also providing a more simplified checklist table for researchers to use. An RT-qPCR
178:
For the process of extracting the DNA/RNA, there are a number of essential guidelines. This includes a description of the extraction process done, a statement on what DNA extraction kit was used and any changes made to the directions, details on whether any
232:
All of the basic information regarding the target is necessary here, including the gene symbol, the accession database number for the sequence in question, the length of the sequence being amplified, information about the specificity screen used such as
303:
process. The only additional desired pieces of information are the chemical composition of the buffer used, who manufactured the plates and tubes used and what their catalog number is, and whether the reaction was set up manually or by a machine.
350:
used or excluded and why, what results were found for the controls with no template genetic material, an explanation for why the reference genes used were chosen and why the number of them was chosen, the method used to
86:
for the broader form of the guidelines. Other researchers have been creating further versions for specific forms of qPCR that may require a supplementary or different set of items to check, including
79:
were set up as a sort of checklist for each step of the procedure with certain items being marked as essential (E) when submitting data for publication and others marked as just desirable (D).
208:
The primary essential parts for this phase include detailing the reaction conditions in full, giving both the amount of RNA used and the total volume of the reaction, give information on the
299:
was used and its concentration, what kit was used and its manufacturer, what additives to the reaction were used, who manufactured the qPCR machine, and what parameters were set for the
698:
Huggett JF, Foy CA, Benes V, Emslie K, Garson JA, Haynes R, Hellemans J, Kubista M, Mueller RD, Nolan T, Pfaffl MW, Shipley GL, Vandesompele J, Wittwer CT, Bustin SA (June 2013).
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used, and lastly the temperature and amount of time done for the reaction. It is also desirable to have the catalog numbers of reagents used and their manufacturers, the
779:
326:
squared) for the calibration curves, the dynamic range of the linear curves, the Cq found at the lowest concentration where 95% of the results were still positive (
196:. Four desired pieces of information are where the reagents used were obtained from, what level of genetic purity was obtained, what yield was obtained, and an
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It is also desired to include information on the number of biological replicates and whether they matched the results from the technical replicates, the
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was developed alongside
Stephen Bustin using the MIQE guidelines for clinical biomarkers in December 2020 in order to identify the clinical presence of
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The MIQE guidelines were created due to the low quality of qPCR data submitted to academic journals at the time, which was only becoming more common as
82:
An additional version of the guidelines was published in
September 2010 for use with fluorescence-based quantitative real-time PCR. It also acted as a
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Bustin SA, Benes V, Garson JA, Hellemans J, Huggett J, Kubista M, Mueller R, Nolan T, Pfaffl MW, Shipley GL, Vandesompele J, Wittwer CT (April 2009).
856:"Droplet Digital PCR versus qPCR for gene expression analysis with low abundant targets: from variable nonsense to publication quality data"
31:
experiments and data, as devised by Bustin et al. in 2009. They were devised after a paper was published in 2002 that claimed to detect
28:
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610:"MIQE précis: Practical implementation of minimum standard guidelines for fluorescence-based quantitative real-time PCR experiments"
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for each primer is. There are several desired, but not required information pieces for this section, such as the location of the
471:
741:
Houreld NN (February 2017). "Are MIQE Guidelines Being
Adhered to in qPCR Investigations in Photobiomodulation Experiments?".
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94:(dPCR). Appropriate adherence to the existing MIQE guidelines has also been overviewed in other scientific areas, including
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Ruijter JM, Lefever S, Anckaert J, Hellemans J, Pfaffl MW, Benes V, Bustin SA, Vandesompele J, Untergasser A (June 2015).
1034:"The Digital MIQE Guidelines Update: Minimum Information for Publication of Quantitative Digital PCR Experiments for 2020"
663:
914:
813:"Talking the talk, but not walking the walk: RT-qPCR as a paradigm for the lack of reproducibility in molecular research"
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to create the MIQE guidelines to provide a baseline level of quality for qPCR data published in scientific literature.
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The extra desired information includes evidence given that qPCR optimization occurred by the use of gradients, the
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1094:"CoV2-ID, a MIQE-compliant sub-20-min 5-plex RT-PCR assay targeting SARS-CoV-2 for the diagnosis of COVID-19"
700:"The Digital MIQE Guidelines: Minimum Information for Publication of Quantitative Digital PCR Experiments"
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Bustin SA, Beaulieu JF, Huggett J, Jaggi R, Kibenge FS, Olsvik PA, Penning LC, Toegel S (September 2010).
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Stahlberg A, Kubista M (February 2018). "Technical aspects and recommendations for single-cell qPCR".
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419:"The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments"
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were included, how repeatable was the data within the assays, what methods were used to determine
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Shipley, Greg (2011). "The MIQE Guidelines
Uncloaked". In Kennedy, Suzanne; Oswald, Nick (eds.).
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to show efficiency of the qPCR, and the confidence intervals for the entire range tested.
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of the results, and what software was used for this part of the qualitative analysis.
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analysis software used and also submitting the raw data to the relevant databases.
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When setting up their new comparative qPCR systems titled "Dots in Boxes" in 2017,
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that allows for active marking of the MIQE checklist as each step is completed.
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Minimum
Information for Publication of Quantitative Real-Time PCR Experiments
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In 2009, Stephen Bustin led an international group of scientists including
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for the Cq with and without the transcriptase being involved, and how the
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to know the volume or mass of the sample that was processed for the qPCR.
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is used, then the efficiency and LOD must be given for each assay done.
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RNA Methodologies: Laboratory Guide for
Isolation and Characterization
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used as a primer and its concentration, the concentration and type of
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Bustin S, Coward A, Sadler G, Teare L, Nolan T (December 17, 2020).
986:"The MIQE Guidelines' tenth anniversary: The good and bad students"
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132:
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Abdel Nour AM, Azhar E, Damanhouri G, Bustin SA (February 2014).
27:) guidelines are a set of protocols for conducting and reporting
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Riedmaier I, Spornraft M, Kirchner B, Pfaffl MV (January 2016).
242:
221:
518:"Five years MIQE guidelines: the case of the Arabian countries"
1186:"RDML-Ninja and RDMLdb for standardized exchange of qPCR data"
330:) along with the evidence for the LOD itself, and lastly if a
467:"The reproducibility of biomedical research: Sleepers awake!"
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was done and the data obtained from it, and any data on the
1244:
577:
Farrell, Robert E. (August 2017). "The MIQE Guidelines".
1158:"Impact of New Technologies in Meeting MIQE Guidelines"
915:"Positioning Digital PCR for Sharper Genomic Views"
369:data for the concentration variants, data on the
854:Taylor SC, Laperriere G, Germain H (May 2017).
241:variants exist for the sequence, and where the
61:with many different languages and protocols.
8:
1163:Genetic Engineering & Biotechnology News
920:Genetic Engineering & Biotechnology News
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818:European Journal of Clinical Investigation
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16:Protocols for reporting qPCR experiments
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913:Marusina, Kate (October 1, 2017).
811:Bustin SA, Nolan T (August 2017).
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1156:Birnie, Andrew (May 14, 2015).
1032:The dMIQE Group (August 2020).
744:Photomedicine and Laser Surgery
785:European Pharmaceutical Review
1:
943:"Guiding our PCR experiments"
664:Molecular Aspects of Medicine
70:Original version developments
1004:10.1016/j.genrep.2020.100630
719:10.1373/clinchem.2013.206375
545:10.1371/journal.pone.0088266
438:10.1373/clinchem.2008.112797
465:Bustin SA (December 2014).
269:of the amplified sequence.
139:viral particles during the
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1121:10.1038/s41598-020-79233-x
882:10.1038/s41598-017-02217-x
54:Next Generation Sequencing
29:quantitative real-time PCR
1286:Polymerase chain reaction
1205:10.1186/s12859-015-0637-6
677:10.1016/j.mam.2017.07.004
485:10.1016/j.bdq.2015.01.002
1249:Horizon Scientific Press
1053:10.1093/clinchem/hvaa125
200:image for confirmation.
110:Tightening of guidelines
629:10.1186/1471-2199-11-74
317:, or from digestion by
228:qPCR target information
174:Nucleic acid extraction
1281:Laboratory techniques
941:Perkel J (May 2015).
757:10.1089/pho.2016.4220
615:BMC Molecular Biology
273:qPCR oligonucleotides
214:reverse transcriptase
204:Reverse transcription
1251:. pp. 151–166.
587:. pp. 287–288.
356:technical replicates
339:confidence intervals
313:material, showing a
190:quantification cycle
59:developing countries
1112:2020NatSR..1022214B
874:2017NatSR...7.2409T
536:2014PLoSO...988266A
354:the data, how many
278:sequences from the
267:secondary structure
198:electrophoresis gel
156:Experimental design
147:Guidelines overview
116:New England Biolabs
100:clinical biomarkers
1191:BMC Bioinformatics
1099:Scientific Reports
1039:Clinical Chemistry
861:Scientific Reports
705:Clinical Chemistry
424:Clinical Chemistry
319:restriction enzyme
263:sequence alignment
218:standard deviation
96:photobiomodulation
1276:Molecular biology
962:10.2144/000114283
832:10.1111/eci.12801
261:exist, whether a
141:COVID-19 pandemic
35:in children with
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255:pseudogenes
123:creating a
92:digital PCR
1270:Categories
998:: 100630.
792:(6): 32–36
377:References
297:polymerase
194:inhibitors
125:mobile app
1012:212994609
671:: 28–35.
479:: 35–42.
352:normalize
332:multiplex
1224:26087842
1169:June 13,
1140:33335187
1072:32746458
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1131:7747624
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