The DOI matches its Crossref record; 92 of the 92 references it cites were found in a registry or an index; 19 of the 46 sources its claims cite were checked for support.
The MIQE Guidelines: Minimum Information for Publication of Quantitative Real-Time PCR Experiments
Open accessAbstract
Background: Currently, a lack of consensus exists on how best to perform and interpret quantitative real-time PCR (qPCR) experiments. The problem is exacerbated by a lack of sufficient experimental detail in many publications, which impedes a reader’s ability to evaluate critically the quality of the results presented or to repeat the experiments. Content: The Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines target the reliability of results to help ensure the integrity of the scientific literature, promote consistency between laboratories, and increase experimental transparency. MIQE is a set of guidelines that describe the minimum information necessary for evaluating qPCR experiments. Included is a checklist to accompany the initial submission of a manuscript to the publisher. By providing all relevant experimental conditions and assay characteristics, reviewers can assess the validity of the protocols used. Full disclosure of all reagents, sequences, and analysis methods is necessary to enable other investigators to reproduce results. MIQE details should be published either in abbreviated form or as an online supplement. Summary: Following these guidelines will encourage better experimental practice, allowing more reliable and unequivocal interpretation of qPCR results.
Overview
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Claims the paper makes
19 Own interpretation · 11 No full text of the sources · 8 Supported · 7 Sources not found · 6 Partly supported · 2 Supported so far · 1 No basis given · accuracy not yet measured
The sources with a verdict support this claim; the rest weren’t checked yet, have no full text we could read, or their verdicts aren’t rated yet.
In the paper
“If multiple reactions are performed, however, accurate quantification of lower concentrations can be obtained via digital PCR (29,63,64 )”
§ The MIQE Guidelines: Minimum Information for Publication of Quantitative Real-Time PCR Experiments > 7.4.3. LOD.
What the sources say
- Not checked — no full text of the sourcecites [29] 2008, Nat Protoc source
- Supportedcites [63] 2008, PLoS ONE source
The Results section explains that digital PCR partitions a sample into hundreds of reaction chambers. At low target concentrations, counting positive chambers closely approximates the true concentration, supporting the claim that multiple reactions enable accurate quantification at lower concentrations. (LLM, conf=0.92)
- Supportedcites [64] 1999, Proc Natl Acad Sci U S A source
The Discussion (page not provided) says digital PCR can detect mutations at relatively low levels and that its detection limit depends on the number of wells analyzed. It also describes quantitative analysis of PCR products, supporting the claim that multiple reactions enable quantification at low levels; the paper’s example concerns rare variants within a sample. (LLM, conf=0.86)
plainstates a cause- Asserted: passes — The authors put it forward as true.
- Checkable: passes — It can be checked against evidence.
- Load-bearing: unclear — It is unclear whether the paper’s conclusions depend on it.
- Based: passes — The paper gives a reason why it holds.
Every source the paper cites for this claim was checked and supports it.
In the paper
“Consequently, many molecular analyses still contain qPCR data that are poorly normalized.”
§ The MIQE Guidelines: Minimum Information for Publication of Quantitative Real-Time PCR Experiments > 8.1. NORMALIZATION
What the sources say
- Supportedcites [70] 2008, Plant Biotechnol J source
The Introduction (page not provided) reports that more than 95% of RT-PCR analyses surveyed in three plant journals used improperly validated reference genes. This supports the claim that many analyses contained qPCR data at risk of poor normalization, though the paper’s evidence concerns plant research. (LLM, conf=0.91)
plain- Asserted: passes — The authors put it forward as true.
- Checkable: passes — It can be checked against evidence.
- Load-bearing: passes — The paper’s conclusions depend on it.
- Based: passes — The paper gives a reason why it holds.
- Supported
The cited sources support only part of this claim — read each source’s verdict below.
In the paper
“which is particularly true for mRNAs that specify proteins that are part of multifunction protein complexes (37 ).”
§ The MIQE Guidelines: Minimum Information for Publication of Quantitative Real-Time PCR Experiments > 2. Conceptual Considerations
What the sources say
- Partly supportedcites [37] 2007, Mol Syst Biol sourceExperimental — accuracy not yet measured
The Functional pathway analysis reports very low or negative protein–mRNA correlations for most multisubunit protein complexes, supporting the claim of poor concordance for their subunits. It does not establish that these complexes are multifunctional; that qualifier differs from the paper’s “multisubunit.” (LLM, conf=0.82)
plainstates an association- Asserted: passes — The authors put it forward as true.
- Checkable: passes — It can be checked against evidence.
- Load-bearing: unclear — It is unclear whether the paper’s conclusions depend on it.
- Based: passes — The paper gives a reason why it holds.
- Partly supported
The sources with a verdict support this claim; the rest weren’t checked yet, have no full text we could read, or their verdicts aren’t rated yet.
In the paper
“these splicing variants specify alternative protein isoforms”
§ The MIQE Guidelines: Minimum Information for Publication of Quantitative Real-Time PCR Experiments > 2. Conceptual Considerations
What the sources say
- Supportedcites [31] 2007, BMC Bioinformatics source
The Background states that a given transcript can be predicted to produce a particular protein isoform, supporting the claim that splicing variants specify alternative isoforms. It also cautions that a transcript may not actually be translated, a distinction the claim does not rule out. (LLM, conf=0.90)
- Not checked — no full text of the sourcecites [32] 2008, J Biol Chem source
plainstates a cause- Asserted: passes — The authors put it forward as true.
- Checkable: passes — It can be checked against evidence.
- Load-bearing: passes — The paper’s conclusions depend on it.
- Based: passes — The paper gives a reason why it holds.
- Supported
Every source the paper cites for this claim was checked and supports it.
In the paper
“a systematic evaluation has revealed that qPCR data-analysis methods differ substantially in their performance (68 )”
§ The MIQE Guidelines: Minimum Information for Publication of Quantitative Real-Time PCR Experiments > 8. Data Analysis
What the sources say
- Supportedcites [68] 2007, BMC Bioinformatics source
The Abstract Conclusion states that qPCR data-analysis methods differ significantly in performance. The Discussion’s evaluation of calculation models identifies differences in resolution, precision, and robustness, supporting the citing sentence’s summary. (LLM, conf=0.95)
plain- Asserted: passes — The authors put it forward as true.
- Checkable: passes — It can be checked against evidence.
- Load-bearing: passes — The paper’s conclusions depend on it.
- Based: passes — The paper gives a reason why it holds.
- Supported
The cited sources support only part of this claim — read each source’s verdict below.
In the paper
“with variation in splicing patterns reported in different tissues or at different developmental stages.”
§ The MIQE Guidelines: Minimum Information for Publication of Quantitative Real-Time PCR Experiments > 2. Conceptual Considerations
What the sources say
- Partly supportedcites [31] 2007, BMC Bioinformatics sourceExperimental — accuracy not yet measured
The Utility section describes an exon occurring specifically in muscular, and possibly nervous, tissue; the Construction and content section also describes charts of tissue distribution for splicing events. These passages support variation across tissues, but our copy does not report variation across developmental stages (searched: developmental stages, developmentally regulated splicing). No page numbers are available in the indexed passages. (LLM, conf=0.85)
- Not checked — no full text of the sourcecites [32] 2008, J Biol Chem source
plainstates an association- Asserted: passes — The authors put it forward as true.
- Checkable: passes — It can be checked against evidence.
- Load-bearing: passes — The paper’s conclusions depend on it.
- Based: passes — The paper gives a reason why it holds.
- Partly supported
Every source the paper cites for this claim was checked and supports it.
In the paper
“no inhibitors copurified with the DNA will distort results, e.g., pathogen detection and their quantification (55 ).”
§ The MIQE Guidelines: Minimum Information for Publication of Quantitative Real-Time PCR Experiments > 5.2. DNA SAMPLES
What the sources say
- Supportedcites [55] 2005, J Virol Methods source
The Discussion says the internal control reveals PCR inhibitors, while the Abstract says it prevents false-negative results and under-reporting caused by problems with PCR amplification. Together, these passages support the claim that inhibitors carried through DNA preparation can distort pathogen detection and quantification. (LLM, conf=0.90)
plainstates a cause- Asserted: passes — The authors put it forward as true.
- Checkable: passes — It can be checked against evidence.
- Load-bearing: passes — The paper’s conclusions depend on it.
- Based: passes — The paper gives a reason why it holds.
- Supported
Every source the paper cites for this claim was checked and supports it.
In the paper
“different PCR reactions may not be equally susceptible to inhibition by substances copurified in nucleic acid extracts (56,57 ).”
§ The MIQE Guidelines: Minimum Information for Publication of Quantitative Real-Time PCR Experiments > 5.2. DNA SAMPLES
What the sources say
- Supportedcites [56] 2008, BMC Res Notes source
The Discussion states that inhibitors copurified during nucleic acid extraction can affect different PCR reactions differently. This directly supports the citing sentence. (LLM, conf=0.95)
- Supportedcites [57] 2003, Clin Chem source
The discussion on p. 8 says that inhibitory components in biological samples need not affect all PCRs equally. It also reports a patient sample in which the IgL λ reaction was strongly inhibited while the IgL κ reaction retained its expected efficiency. (LLM, conf=0.94)
hedgedstates a possible cause- Asserted: passes — The authors put it forward as true.
- Checkable: passes — It can be checked against evidence.
- Load-bearing: passes — The paper’s conclusions depend on it.
- Based: passes — The paper gives a reason why it holds.
- Supported
Every source the paper cites for this claim was checked and supports it.
In the paper
“variations in tissue-preparation procedures have a substantial effect on both RNA yield and quality (47 ).”
§ The MIQE Guidelines: Minimum Information for Publication of Quantitative Real-Time PCR Experiments > 4. Sample Acquisition, Handling, and Preparation
What the sources say
- Supportedcites [47] 2007, Biotechniques source
The passage labeled References (page not provided) explicitly says that variations in tissue preparation affected both RNA quality and quantity. It reports RNA yields of 28.3%–97.1% of expected and poststaining quality scores of 2.3–7.9 across protocols. (LLM, conf=0.94)
plainstates a cause- Asserted: passes — The authors put it forward as true.
- Checkable: passes — It can be checked against evidence.
- Load-bearing: passes — The paper’s conclusions depend on it.
- Based: passes — The paper gives a reason why it holds.
- Supported
Every source the paper cites for this claim was checked and supports it.
In the paper
“autosomal nonimprinted genes that display allelic imbalance in their expression have been described (33 ).”
§ The MIQE Guidelines: Minimum Information for Publication of Quantitative Real-Time PCR Experiments > 2. Conceptual Considerations
What the sources say
- Supportedcites [33] 2008, Genome Res source
The Discussion reports two nonimprinted genes with allele-specific expression imbalance under sequence-dependent cis regulation. The parent-of-origin analysis gives ERAP2 as an example: its allelic skewing depended on the C allele, not parental origin. (LLM, conf=0.94)
plain- Asserted: passes — The authors put it forward as true.
- Checkable: passes — It can be checked against evidence.
- Load-bearing: passes — The paper’s conclusions depend on it.
- Based: passes — The paper gives a reason why it holds.
- Supported
- It may state two separate claims as one. 12
- Probably not a substantial claim. 6
The cited sources support only part of this claim — read each source’s verdict below.
In the paper
“only a couple of interlaboratory comparisons have been performed, and both of these studies emphasized the need for standardization of qPCR diagnostic assays (44,45 ).”
§ The MIQE Guidelines: Minimum Information for Publication of Quantitative Real-Time PCR Experiments > 3. Research vs Diagnostic Applications
What the sources say
- Not checked — no full text of the sourcecites [44] 2006, Clin Chem source
- Partly supportedcites [45] 2008, J Clin Microbiol sourceExperimental — accuracy not yet measured
The discussion (p. 4) describes this study as the first international evaluation of HIV-2 RNA quantification assays and says the collaboration is working toward standardization because results varied between laboratories. It does not establish that only a couple of interlaboratory comparisons of qPCR diagnostic assays had been performed generally. (LLM, conf=0.85)
plainstates an association- Asserted: passes — The authors put it forward as true.
- Checkable: passes — It can be checked against evidence.
- Load-bearing: unclear — It is unclear whether the paper’s conclusions depend on it.
- Based: passes — The paper gives a reason why it holds.
It may state two separate claims as one.
Every source the paper cites for this claim was checked and supports it.
In the paper
“although qPCR has frequently been proclaimed a touchstone or a gold standard, in practice this “standard” is a variable one, and the reporting of results requires considerable sophistication of analysis and interpretation (43 ).”
§ The MIQE Guidelines: Minimum Information for Publication of Quantitative Real-Time PCR Experiments > 2. Conceptual Considerations
What the sources say
- Supportedcites [43] 2005, J Mol Endocrinol source
The Introduction says real-time RT-PCR is not an established, standardised technology, while the Normalisation section describes variable results and multiple ways of presenting data. The Conclusion calls the results subjective and urges guidelines for their analysis and reporting. Together, these passages support the claim's characterization of qPCR as a variable standard requiring careful interpretation. (LLM, conf=0.90)
plain- Asserted: passes — The authors put it forward as true.
- Checkable: passes — It can be checked against evidence.
- Load-bearing: passes — The paper’s conclusions depend on it.
- Based: passes — The paper gives a reason why it holds.
It may state two separate claims as one.
- Rests on an assumption: A large part of the variance in reported RT-qPCR expression values comes from data-processing algorithm corrections rather than solely from protocol variation.
- Supported
The cited sources support only part of this claim — read each source’s verdict below.
In the paper
“The transcripts of most human genes and many genes in other multicellular organisms are alternatively spliced (31,32 ),”
§ The MIQE Guidelines: Minimum Information for Publication of Quantitative Real-Time PCR Experiments > 2. Conceptual Considerations
What the sources say
- Partly supportedcites [31] 2007, BMC Bioinformatics sourceExperimental — accuracy not yet measured
The Utility section reports that 61% of the human genes in the database were estimated to undergo alternative splicing, supporting the claim about most human genes. The passages found do not establish the claim about genes in other multicellular organisms; the paper’s analysis here concerns human genes. (LLM, conf=0.86)
- Not checked — no full text of the sourcecites [32] 2008, J Biol Chem source
plain- Asserted: passes — The authors put it forward as true.
- Checkable: passes — It can be checked against evidence.
- Load-bearing: passes — The paper’s conclusions depend on it.
- Based: passes — The paper gives a reason why it holds.
It may state two separate claims as one.
- Partly supported
The cited sources support only part of this claim — read each source’s verdict below.
In the paper
“Unfortunately, although there is an increased awareness of the importance of systematic validation and although the potentially highly misleading effects of the use of inappropriate reference genes for normalization are widely known, these considerations also are still widely disregarded (70 ).”
§ The MIQE Guidelines: Minimum Information for Publication of Quantitative Real-Time PCR Experiments > 8.1. NORMALIZATION
What the sources say
- Partly supportedcites [70] 2008, Plant Biotechnol J sourceExperimental — accuracy not yet measured
The Introduction (page not provided) says that improperly validated reference genes remained widely used in plant RT-PCR and that their use may lead to misinterpretation; the paper also demonstrates a misleading normalization result. But it says the importance of earlier findings did not appear to have been recognized, rather than establishing increased awareness or that the risks were widely known. (LLM, conf=0.86)
boosted- Asserted: passes — The authors put it forward as true.
- Checkable: passes — It can be checked against evidence.
- Load-bearing: passes — The paper’s conclusions depend on it.
- Based: passes — The paper gives a reason why it holds.
It may state two separate claims as one.
- Partly supported
Every source the paper cites for this claim was checked and supports it.
In the paper
“alternative, more generalized quantitative models have been developed to correct for differences in amplification efficiency (62 ) and to allow the use of multiple reference genes (30 ).”
§ The MIQE Guidelines: Minimum Information for Publication of Quantitative Real-Time PCR Experiments > 7.4.1. PCR efficiency.
What the sources say
- Supportedcites [62] 2001, Nucleic Acids Res source
The citation to Pfaffl is attached to the claim about correcting for differences in amplification efficiency, not the separate claim about multiple reference genes. In “Mathematical model for relative quantification in realtime PCR” (page unavailable), the model uses individual efficiencies for the target and reference transcripts; the Discussion contrasts it with a method that presumes identical efficiencies. (LLM, conf=0.92)
- Supportedcites [30] 2007, Genome Biol source
The citation to this source is attached to the use of multiple reference genes. In Results and discussion (§ qBase), the paper describes its generalized model as providing multiple-reference-gene normalization; the Conclusion also states that the model allows normalization with multiple reference genes. (LLM, conf=0.94)
plainstates a cause- Asserted: passes — The authors put it forward as true.
- Checkable: passes — It can be checked against evidence.
- Load-bearing: unclear — It is unclear whether the paper’s conclusions depend on it.
- Based: passes — The paper gives a reason why it holds.
It may state two separate claims as one.
- Supported
The cited sources support only part of this claim — read each source’s verdict below.
In the paper
“The necessity for ensuring quality-assurance measures for qPCR and RT-qPCR assays is well recognized (25,44,(75)(76)(77)(78)(79)(80)(81)(82)(83)(84)(85)(86).”
§ The MIQE Guidelines: Minimum Information for Publication of Quantitative Real-Time PCR Experiments > Conclusions
What the sources say
- Partly supportedcites [25] 2006, BMC Biotechnol sourceExperimental — accuracy not yet measured
The Background (page unavailable) describes measures to improve the reliability of low-level DNA quantification by qPCR. The methods section headed “Reverse primer” (page unavailable) also reports contamination controls and checks on standard-curve quality. These support the need for quality assurance in qPCR, but the paper does not address reverse-transcription qPCR (RT-qPCR). (LLM, conf=0.86)
- Not checked — no full text of the sourcecites [44] 2006, Clin Chem source
- Not checked — no full text of the sourcecites [75] 2005, J Clin Microbiol source
boosted- Asserted: passes — The authors put it forward as true.
- Checkable: passes — It can be checked against evidence.
- Load-bearing: passes — The paper’s conclusions depend on it.
- Based: passes — The paper gives a reason why it holds.
Probably not a substantial claim.
- Partly supported
In the paper
“however, the use of such arbitrary C q cutoffs is not ideal, because they may be either too low (eliminating valid results) or too high (increasing false-positive results) (26 )”
§ The MIQE Guidelines: Minimum Information for Publication of Quantitative Real-Time PCR Experiments > 7.4.1. PCR efficiency.
What the sources say
- Not checked — no full text of the sourcecites [26] 2008, Eur Food Res Technol source
hedgedstates a possible cause- Asserted: passes — The authors put it forward as true.
- Checkable: passes — It can be checked against evidence.
- Load-bearing: unclear — It is unclear whether the paper’s conclusions depend on it.
- Based: passes — The paper gives a reason why it holds.
It may state two separate claims as one.
In the paper
“knowledge of the presence and function of specific microRNAs is as important to understanding gene expression as being able to quantify the mRNA species (38 ).”
§ The MIQE Guidelines: Minimum Information for Publication of Quantitative Real-Time PCR Experiments > 2. Conceptual Considerations
What the sources say
- Not matchedcites [38] 2008, Carcinogenesis
Couldn’t be matched to a paper in Crossref, so it can’t be checked.
boostedstates an association- Asserted: passes — The authors put it forward as true.
- Checkable: fails — It may be too vague to check against evidence.
- Load-bearing: fails — The paper’s conclusions may not depend on it.
- Based: passes — The paper gives a reason why it holds.
Probably not a substantial claim. It may be too vague to check against evidence. The paper’s conclusions may not depend on it.
- Not matched
A conclusion the paper draws from its own results — it cites no other source for it, so there is none to check. The quote shows it is in the paper.
In the paper
“calculations of relative concentrations will be inaccurate, yielding misleading results.”
§ The MIQE Guidelines: Minimum Information for Publication of Quantitative Real-Time PCR Experiments > 7.4.1. PCR efficiency.
plainstates a cause- Asserted: passes — The authors put it forward as true.
- Checkable: passes — It can be checked against evidence.
- Load-bearing: passes — The paper’s conclusions depend on it.
- Based: passes — The paper gives a reason why it holds.
It may state two separate claims as one.
A conclusion the paper draws from its own results — it cites no other source for it, so there is none to check. The quote shows it is in the paper.
In the paper
“single exon-based RT-qPCR assays may detect a number of splice variants, whereas intron-spanning primers may be more selective but may miss some splice variants altogether.”
§ The MIQE Guidelines: Minimum Information for Publication of Quantitative Real-Time PCR Experiments > 2. Conceptual Considerations
hedgedstates a possible cause- Asserted: passes — The authors put it forward as true.
- Checkable: passes — It can be checked against evidence.
- Load-bearing: passes — The paper’s conclusions depend on it.
- Based: passes — The paper gives a reason why it holds.
It may state two separate claims as one.
In the paper
“Use of a 3Ј:5Ј assay requires that the PCR efficiencies of both assays be virtually identical (51 ) and not be subject to differential inhibition.”
§ The MIQE Guidelines: Minimum Information for Publication of Quantitative Real-Time PCR Experiments > 5.1. RNA SAMPLES
What the sources say
- Not checked — no full text of the sourcecites [51] 2006, Nat Protoc source
plain- Asserted: passes — The authors put it forward as true.
- Checkable: passes — It can be checked against evidence.
- Load-bearing: passes — The paper’s conclusions depend on it.
- Based: passes — The paper gives a reason why it holds.
It may state two separate claims as one.
A conclusion the paper draws from its own results — it cites no other source for it, so there is none to check. The quote shows it is in the paper.
In the paper
“Although differences between biological replicates may be large, sufficient numbers may allow smaller experimental differences to be discerned.”
§ The MIQE Guidelines: Minimum Information for Publication of Quantitative Real-Time PCR Experiments > 8.2. VARIABILITY
hedgedstates a possible cause- Asserted: passes — The authors put it forward as true.
- Checkable: passes — It can be checked against evidence.
- Load-bearing: fails — The paper’s conclusions may not depend on it.
- Based: passes — The paper gives a reason why it holds.
Probably not a substantial claim. The paper’s conclusions may not depend on it.
- Rests on an assumption: The inherent variability of biological systems may rival or exceed experimental differences between groups.
A conclusion the paper draws from its own results — it cites no other source for it, so there is none to check. The quote shows it is in the paper.
In the paper
“Thus, the reference genes or materials used for standardization are critical, and any assessment of the validity of an RT-qPCR experiment must also consider the appropriateness of the relative-quantification reference.”
§ The MIQE Guidelines: Minimum Information for Publication of Quantitative Real-Time PCR Experiments > 2. Conceptual Considerations
plainstates an association- Asserted: passes — The authors put it forward as true.
- Checkable: passes — It can be checked against evidence.
- Load-bearing: passes — The paper’s conclusions depend on it.
- Based: passes — The paper gives a reason why it holds.
It may state two separate claims as one.
- Rests on an assumption: Most quantitative RNA measurements are relative, not absolute.
In the paper
“The methods produce different results, making it unwise to try to compare data obtained with the different methods (48 ).”
§ The MIQE Guidelines: Minimum Information for Publication of Quantitative Real-Time PCR Experiments > 5.1. RNA SAMPLES
What the sources say
- Not checked — no full text of the sourcecites [48] 2005, Expert Rev Mol Diagn source
plainstates a cause- Asserted: passes — The authors put it forward as true.
- Checkable: passes — It can be checked against evidence.
- Load-bearing: passes — The paper’s conclusions depend on it.
- Based: passes — The paper gives a reason why it holds.
It may state two separate claims as one.
A conclusion the paper draws from its own results — it cites no other source for it, so there is none to check. The quote shows it is in the paper.
In the paper
“Consequently, power analysis is useful for determining the number of samples necessary for valid conclusions.”
§ The MIQE Guidelines: Minimum Information for Publication of Quantitative Real-Time PCR Experiments > 8.2. VARIABILITY
plainstates a cause- Asserted: passes — The authors put it forward as true.
- Checkable: passes — It can be checked against evidence.
- Load-bearing: fails — The paper’s conclusions may not depend on it.
- Based: passes — The paper gives a reason why it holds.
Probably not a substantial claim. The paper’s conclusions may not depend on it.
- Rests on an assumption: Many factors contribute to experimental variation and influence the number of biological replicates necessary to achieve a given statistical power.
A conclusion the paper draws from its own results — it cites no other source for it, so there is none to check. The quote shows it is in the paper.
In the paper
“This difference must be clearly recognized, and one cannot assume that legacy PCR assays can translate directly into the qPCR format.”
§ The MIQE Guidelines: Minimum Information for Publication of Quantitative Real-Time PCR Experiments > Conclusions
plain- Asserted: passes — The authors put it forward as true.
- Checkable: passes — It can be checked against evidence.
- Load-bearing: passes — The paper’s conclusions depend on it.
- Based: passes — The paper gives a reason why it holds.
Probably not a substantial claim.
- Rests on an assumption: The main difference between qPCR and conventional PCR is the expectation that qPCR can quantify target nucleic acids accurately.
A conclusion the paper draws from its own results — it cites no other source for it, so there is none to check. The quote shows it is in the paper.
In the paper
“As a consequence, investigations that use this widely applied technology will produce data that are more uniform, more comparable, and, ultimately, more reliable.”
§ The MIQE Guidelines: Minimum Information for Publication of Quantitative Real-Time PCR Experiments > Conclusions
plainstates a cause- Asserted: passes — The authors put it forward as true.
- Checkable: fails — It may be too vague to check against evidence.
- Load-bearing: passes — The paper’s conclusions depend on it.
- Based: fails — The paper may give no reason why it holds.
Probably not a substantial claim. It may be too vague to check against evidence. The paper may give no reason why it holds. It may state two separate claims as one.
The paper relies on this without support.
Many qPCR publications lack enough experimental detail for readers to assess or repeat the work.
“The problem is exacerbated by a lack of sufficient experimental detail in many publications, which impedes a reader's ability to evaluate critically the quality of the results presented or to repeat the experiments.”
“The problem is exacerbated by the lack of information that characterizes most reports of studies that have used this technology, with many publications not providing sufficient experimental detail to permit the reader to critically evaluate the quality of the results presented or to repeat the experiments.”
The paper relies on this without support.
Presence of an mRNA does not establish that it is translated into protein or functional protein.
“detection of the presence of an mRNA provides no information on whether that mRNA will be translated into a protein or, indeed, whether a functional protein is translated at all.”
The paper relies on this without support.
Most quantitative RNA measurements are relative, not absolute.
“most quantitative RNA data are not absolute, but relative.”
The paper relies on this without support.
A large part of the variance in reported RT-qPCR expression values comes from data-processing algorithm corrections rather than solely from protocol variation.
“Much of the variance in reported expression values produced in RT-qPCR experiments is not simply due to variation in experimental protocols but is caused by corrections applied by various dataprocessing algorithms, each of which makes its own assumptions about the data.”
The paper relies on this without support.
PCR efficiency is highly dependent on the primers used.
“PCR efficiency is highly dependent on the primers used”
The paper relies on this without support.
The structure of the nucleic acid target has a substantial impact on the efficiency of reverse transcription and PCR.
“The structure of the nucleic acid target (e.g., stem and loop secondary RNA structure) has a substantial impact on the efficiency of reverse transcription and the PCR.”
The paper relies on this without support.
A marked presence of primer dimers produces lower PCR efficiency in probe-based assays and may generate false positives in SYBR Green I assays.
“A marked presence of primer dimers produces a lower PCR efficiency in probe-based assays and may generate false positives in assays based on SYBR Green I.”
The paper relies on this without support.
No-template controls detect PCR contamination when probes are used and can distinguish unintended amplification products from intended products in SYBR Green I reactions.
“NTCs detect PCR contamination when probes are used and can also distinguish unintended amplification products (e.g., primer dimers) from the intended PCR products in SYBR Green I reactions.”
The paper relies on this without support.
Robust and precise qPCR assays are usually correlated with high PCR efficiency.
“Robust and precise qPCR assays are usually correlated with high PCR efficiency.”
The paper relies on this without support.
PCR efficiency is particularly important when reporting mRNA concentrations for target genes relative to reference genes.
“PCR efficiency is particularly important when reporting mRNA concentrations for target genes relative to those of reference genes.”
The paper relies on this without support.
A260/A280 measurement is not sufficient for quantitative nucleic acid analysis, especially when measuring minor differences (<10-fold) in cellular mRNA concentrations.
“such measurement is not sufficient if the nucleic acid is to be used for quantitative analysis, especially when the aim is to measure minor differences (Ͻ10-fold) in cellular mRNA concentrations.”
The paper relies on this without support.
RNA integrity numbers relate to rRNA quality and cannot be expected to be an absolute measure of RNA quality.
“these numbers relate to rRNA quality and cannot be expected to be an absolute measure of quality.”
The paper relies on this without support.
The small amplicon size of qPCR assays helps minimize assay-related problems.
“The small amplicon size of qPCR assays helps to minimize assay-related problems”
The paper relies on this without support.
Lower limits of quantification are usually poorly defined.
“Because lower limits of quantification are usually poorly defined, the variation at the lowest concentration claimed to be within the linear interval should be determined.”
The paper relies on this without support.
Low-copy PCRs are stochastically limited.
“Low-copy PCRs are stochastically limited, and LODs of Ͻ3 copies per PCR are not possible.”
The paper relies on this without support.
Precision in qPCR typically varies with concentration, decreasing with copy number.
“Precision in qPCR typically varies with concentration, decreasing with the copy number.”
The paper relies on this without support.
Use of reference genes as internal controls is the most common method for normalizing cellular mRNA data.
“The use of reference genes as internal controls is the most common method for normalizing cellular mRNA data”
The paper relies on this without support.
The inherent variability of biological systems may rival or exceed experimental differences between groups.
“The inherent variability of biological systems may rival or exceed experimental differences between groups.”
The paper relies on this without support.
Many factors contribute to experimental variation and influence the number of biological replicates necessary to achieve a given statistical power.
“Many factors contribute to experimental variation and influence the number of biological replicates necessary to achieve a given statistical power.”
The paper relies on this without support.
The main difference between qPCR and conventional PCR is the expectation that qPCR can quantify target nucleic acids accurately.
“The main difference between qPCR and conventional (legacy) PCR assays is the expectation of the former's potential to quantify target nucleic acids accurately.”
The paper relies on this without support.
The concern about multiplexing accuracy is particularly important when targets of appreciably lower abundance are coamplified with highly abundant targets.
“This concern is of particular importance when targets of appreciably lower abundance are coamplified with highly abundant targets.”
The paper relies on this without support.
Reference gene utility must be experimentally validated for particular tissues or cell types and specific experimental designs.
“their utility must be experimentally validated for particular tissues or cell types and specific experimental designs”
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“Gene knockdown and PCR techniques rely upon the correct design and experimental use of RNA targeting reagents and PCR primers, respectively [17–19].”
From Semi-automated fact-checking of nucleotide sequence reagents in biomedical research publications: The Seek & Blastn tool (2019) · cites this paper as [18]
Experimental — accuracy not yet measuredThe source backs part of the claim, not all of it — or backs it for a different population, condition or comparison.
In the citing paper
“As the nucleotide sequences of these reagents define their identity and possible experimental use, each published reagent descriptor is recommended to be accompanied by its corresponding nucleotide sequence [18].”
From Semi-automated fact-checking of nucleotide sequence reagents in biomedical research publications: The Seek & Blastn tool (2019) · cites this paper as [18]
Experimental — accuracy not yet measuredThe source backs part of the claim, not all of it — or backs it for a different population, condition or comparison.
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“The Minimum Information for the Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines and related articles published thereafter define a rigorous methodology for designing qPCR experiments that assures publication of reproducible and high quality data5–7.”
From Droplet Digital PCR versus qPCR for gene expression analysis with low abundant targets: from variable nonsense to publication quality data (2017) · cites this paper as [5]
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In the citing paper
“The qPCR method was optimized until the characteristics of the standard curve fitted qPCR standard guidelines32 (amplification efficiency (E) = 100 ± 10% and the coefficient of correlation (R2) > 0.95).”
From An apicomplexan parasite drives the collapse of the bay scallop population in New York (2023) · cites this paper as [32]
Experimental — accuracy not yet measuredThe cited source says what the claim attributes to it.
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“it is also essential that some estimation of efficiency is made (25).”
From Comparison of microfluidic digital PCR and conventional quantitative PCR for measuring copy number variation (2012) · cites this paper as [25]
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“Real-time quantitative PCR was performed in accordance with the MIQE guidelines (Supplementary Table S1 and Supplementary Figure S1) (25).”
From Comparison of microfluidic digital PCR and conventional quantitative PCR for measuring copy number variation (2012) · cites this paper as [25]
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“Quantitative RT-PCR was conducted following the MIQE (minimum information for publication of quantitative real-time PCR experiments) guidelines38.”
From Neurotoxic reactive astrocytes are induced by activated microglia (2017) · cites this paper as [38]
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“Relative expression levels from eight independent experiments were calculated using the quantification cycle (Cq) method, according to MIQE guidelines24.”
From TNF-alpha-induced microglia activation requires miR-342: Impact on NF-kB signaling and neurotoxicity (2020) · cites this paper as [24]
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“TaqMan® miRNA and gene expression assays either in single-tube or array format were designed to follow MIQE (Minimum Information for Publication of Quantitative Real-Time PCR Experiments) guidelines [53] according to the company’s statement.”
From The Mitochondria-Associated ER Membranes Are Novel Subcellular Locations Enriched for Inflammatory-Responsive MicroRNAs (2020) · cites this paper as [53]
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“In line with the MIQE (Minimum Information for Publication of Quantitative Real-Time PCR Experiments), we recommend using at least two housekeeping genes48,49.”
From Algorithmic assessment of cellular senescence in experimental and clinical specimens (2021) · cites this paper as [49]
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“2005, Science, 309, 1694, 10.1126/science.1117768”
Cited work 10.1126/science.1117768 · retracted 2007-04-20 · notice 10.1126/science.316.5823.367b · this paper published 2009-04-01
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Improved taxadiene production by optimizing DXS expression and fusing short-chain prenyltransferases
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“A probability level with a p -value < 0.05 was used as the minimum to indicate the statistical significance.”
MethodInfluentialMaternal high fat–high energy diet alters metabolic factors in the non‐human primate fetal heart
Bertossa, M. R., Darby, J. R. T., Holman, S. L., Meakin, A. S., Li, C., Huber, H. F., Wiese, M. D., Nathanielsz, P. W., & Morrison, J. L. (2024) · The Journal of Physiology · doi:10.1113/jp286861 · Cited by 12
“All qRT-PCR procedures adhered to the MIQE guidelines (Bustin et al., 2009).”
Discovery of a small-molecule inhibitor of KSHV lytic replication from the MMV pandemic response box
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Multifaceted activation of STING axis upon Nipah and measles virus-induced syncytia formation
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“Pfaffl Model [79] and Bustin MIQE checklist [80] were used for all validations and calculations.”
MethodXie, J., Liu, J., Khashaveh, A., et al. (2024) · Journal of Agricultural and Food Chemistry · doi:10.1021/acs.jafc.4c05493 · Cited by 11
Zhang, Y., Wang, X., Wang, X., et al. (2024) · Nano Today · doi:10.1016/j.nantod.2024.102510 · Cited by 11
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“…gene expression using the cDNA template, gene specific primers (Thermo Fisher Custom Primers, Invitrogen, UK – see Table 1) and SYBR green chemistry. qPCR was run as per MIQE guidelines [37], on the Ste-pOne qPCR machine (Applied Biosystems StepOnePlus Real-Time PCR System; StepOne Software v2.”
MethodPrudon, N., Cordero-Espinoza, L., Abarkan, M., Gurchenkov, B., Morel, C., Lepleux, M., Luca, V. D., Lartigue, M., Cabanas, H., Pujol, N., et al. (2024) · Neurotherapeutics · doi:10.1016/j.neurot.2024.e00436 · Cited by 11
Functional analysis of ZmPHR1 and ZmPHR2 under low-phosphate stress in maize
Hu, H., Wang, Y., Zhong, H., et al. (2024) · Molecular Breeding · doi:10.1007/s11032-024-01508-2 · Cited by 11
“For gene expression analysis, RNA isolation, cDNA synthesis and quantitative real-time polymerase chain reaction (RT-qPCR) expression analysis were conducted in accordance with the previously described methodology (Bustin et al. 2009).”
Wang, X., Yang, S., Li, B., et al. (2024) · Plant Cell Reports · doi:10.1007/s00299-024-03352-2 · Cited by 11
“1) and estimated using the 2 −ΔΔCt method (Livak and Schmittgen 2001; Bustin et al. 2009; Wan et al. 2011).”
Li, S., Liu, J., Xu, W., Zhang, S., Zhao, M., Lu, M., Hui, M., Wang, Y., Hou, Y., Cong, B., et al. (2024) · Forensic Science International Genetics · doi:10.1016/j.fsigen.2024.103180 · Cited by 14
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Changes in mucosal epithelia of marine Atlantic salmon (Salmo salar) under chronic heat stress
Sharifian, M., English, C. J., Whitehead, D. L., et al. (2024) · Aquaculture · doi:10.1016/j.aquaculture.2024.742064 · Cited by 11
Persistence of Reptile DNA in a Terrestrial Substrate: A Case Study Using the Eastern Indigo Snake
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“Assuming three replicates and 5% false negative rate (Bustin et al. 2009), the LOD is 1.56.”
InfluentialKhan, F., Esmaeily, M., Jin, G., Sevin, S., Jung, C., Ham, E., & Kim, Y. (2025) · Pesticide Biochemistry and Physiology · doi:10.1016/j.pestbp.2025.106331 · Cited by 13
qPCR-Based Molecular Detection of Trichophyton indotineae by Targeting Divergent Sequences
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“Genomic DNA from T . indotineae (CBS 149165) was tested using analytical parameters such as slope, efficiency, linearity, repeatability, and detection limit, in accordance with the Minimum Information for Pub-lication of Quantitative Real-Time PCR Experiments guidelines [23].”
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“Relative expression levels were calculated using the quantification cycle (Ct) method, according to Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines [34].”
Characterization of LEDGF/p75 Genetic Variants and Association with HIV-1 Disease Progression
Messiaen, P., Spiegelaere, W. D., Alcamı́, J., Vervisch, K., Acker, P. V., Verhasselt, B., Meuwissen, P., Calonge, E., Gonzàlez, N., Gutiérrez, F., et al. (2012) · PLoS ONE · doi:10.1371/journal.pone.0050204 · Cited by 11
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Self-citationIdentification of lactic acid bacteria isolated from wine using real-time PCR
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Detection of Two Peronospora spp., Responsible for Downy Mildew, in Opium Poppy Seed
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“In the prfA assay, the AmpliTaq Gold and Hot Start Taq failed completely (“no Cq”; Cq= quantification cycle: cycle that reaches the fluorescence threshold [16]) and results of the Maxima Probe/ROX qPCR Master Mix were problematic because respective Cq-values were all above 37 and incorrectly ranged (Fig.”
BackgroundMethodInfluentialSpecific and reliable detection of Myosin 1C isoform A by RTqPCR in prostate cancer cells
Saidova, A. A., Potashnikova, D. M., Tvorogova, A. V., et al. (2018) · PeerJ · doi:10.7717/peerj.5970 · Cited by 11
“In all cases one has to follow the MIQE guidelines that describe the minimum information for publication of PCRdata to provide the consistency between the laboratories and reproducibility of clinical and scientific results (Bustin et al., 2009).”
MethodZHENG, L.-Y., Zhang, Z., ZHANG, J.-M., et al. (2019) · Journal of Integrative Agriculture · doi:10.1016/s2095-3119(18)61973-2 · Cited by 11
Szenteczki, M. A., Pitteloud, C., Casacci, L. P., et al. (2019) · Ecology and Evolution · doi:10.1002/ece3.5010 · Cited by 11
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siRNA Knockdown of Mammalian zDHHCs and Validation of mRNA Expression by RT-qPCR
McClafferty, H., & Shipston, M. J. (2019) · Methods in molecular biology · doi:10.1007/978-1-4939-9532-5_12 · Cited by 11
Omeka, W. K. M., Liyanage, D. S., Yang, H., & Lee, J. (2019) · Fish & Shellfish Immunology · doi:10.1016/j.fsi.2019.09.071 · Cited by 11
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Brito, L. F., Schultenkämper, K., Passaglia, L. M. P., et al. (2020) · Applied Microbiology and Biotechnology · doi:10.1007/s00253-020-10571-6 · Cited by 11
“Effect of CRISPRi on biofilm formation by P. sonchi Spo0A regulates biofilm development in B. subtilis and B. methanolicus (Hamon and Lazazzera 2001; Bustin et al. 2009; Schultenkämper et al. 2019).”
BackgroundMethodHojny, J., Bártů, M., Krkavcová, E., Němejcová, K., Sevcik, J., Cibula, D., Frýba, V., Plincelnerova, L., Dundr, P., & Stružinská, I. (2020) · Scientific Reports · doi:10.1038/s41598-020-63733-x · Cited by 11
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Hampton, T. H., Koeppen, K., Bashor, L., et al. (2020) · American Journal of Physiology-Lung Cellular and Molecular Physiology · doi:10.1152/ajplung.00158.2020 · Cited by 11
A Combined RNA Preservation and Extraction Protocol for Gene Expression Studies in Cacao Beans
Wever, J. D., Tulkens, D., Verwaeren, J., et al. (2020) · Frontiers in Plant Science · doi:10.3389/fpls.2020.00992 · Cited by 11
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Mariot, V., Joubert, R., Marsollier, A.-C., Hourdé, C., Voït, T., & Dumonceaux, J. (2020) · Molecular Therapy — Nucleic Acids · doi:10.1016/j.omtn.2020.10.028 · Cited by 11
“The qPCR Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) standards were followed.(44) To determine the best reference gene in vivo, six different reference genes (Gapdh, Gus, Actin, Hprt1, Cyclophilin, and Psma2) were tested and Gapdh was validated as a reference gene for which Ct…”
MethodOfiteru, A., Becheru, D. F., Gharbia, S., Baltă, C., Herman, H., Mladin, B., Ioniţă, M., Hermenean, A. O., & Burns, J. S. (2020) · Cells · doi:10.3390/cells9122559 · Cited by 10
“The MIQE guidelines discouraged large discrepancies in transcript abundance between reference and target genes and recommend more than one reference gene [71].”
BackgroundPan, A. V., Kuhn, C., Schmolz, E., von Samson‐Himmelstjerna, G., & Krücken, J. (2020) · International Journal for Parasitology Drugs and Drug Resistance · doi:10.1016/j.ijpddr.2020.11.003 · Cited by 12
“At the end of every extension phase, the amount of double stranded PCR product was measured as BRYT Green® fluorescence, and the quantification cycle (Cq) was determined according to the MIQE guidelines (Bustin et al., 2009).”
MethodThe Gustavus Gene Can Regulate the Fecundity of the Green Peach Aphid, Myzus persicae (Sulzer)
Gao, Y., Ren, R., Peng, J. Y., et al. (2021) · Frontiers in Physiology · doi:10.3389/fphys.2020.596392 · Cited by 11
“According to Bustin et al. (2009), the relevant information for PCR primer verification is provided in Supplementary Figures S1, S2.”
BackgroundCosta, E. D., Dai, F., Lecchi, C., et al. (2021) · Veterinary Record · doi:10.1002/vetr.82 · Cited by 11
“The RT-qPCR experiments 107 were designed following MIQE guidelines (26).”
MethodBurman, R. J., Watson, L. M., Smith, D. C., Raimondo, J. V., Ballo, R., Scholefield, J., Cowley, S. A., Wood, M. J. A., Kidson, S. H., & Greenberg, L. (2021) · PLoS ONE · doi:10.1371/journal.pone.0247434 · Cited by 11
Maurya, G. K., Chaudhary, R., Pandey, N., & Misra, H. S. (2021) · Journal of Biological Chemistry · doi:10.1016/j.jbc.2021.100451 · Cited by 11
Drzewiecka, E. M., Kozlowska, W., Żmijewska, A., Wydorski, P. J., & Franczak, A. (2021) · International Journal of Molecular Sciences · doi:10.3390/ijms22062920 · Cited by 11
“The Real-Time PCR was performed according to the guidelines provided by Bustin et al. (2009) [58].”
MethodFerreira, M. M., Mechler-Dreibi, M. L., Sonálio, K., et al. (2021) · Veterinary Microbiology · doi:10.1016/j.vetmic.2021.109123 · Cited by 11
Staege, S., Kutschenko, A., Baumann, H., Glaß, H., Henkel, L. M., Gschwendtberger, T., Kalmbach, N., Klietz, M., Hermann, A., Lohmann, K., et al. (2021) · Frontiers in Cell and Developmental Biology · doi:10.3389/fcell.2021.650586 · Cited by 11
Innate Antiviral Cytokine Response to Swine Influenza Virus by Swine Respiratory Epithelial Cells
Bakre, A. A., Jones, L. P., Murray, J., Reneer, Z. B., Meliopoulos, V., Cherry, S., Schultz‐Cherry, S., & Tripp, R. A. (2021) · Journal of Virology · doi:10.1128/jvi.00692-21 · Cited by 11
“All qPCR assay designs were as per MIQE 2 guidelines (71, 72).”
MethodZeng, J., Mu, L.-L., Jin, L., Anjum, A. A., & Li, G. (2021) · Bulletin of Entomological Research · doi:10.1017/s0007485321000420 · Cited by 12
“According to a previously described method (Bustin et al., 2009), the generation of specific PCR products was confirmed by gel electrophoresis.”
MethodRizzo, D., Lio, D. D., Bartolini, L., et al. (2021) · Journal of Economic Entomology · doi:10.1093/jee/toab138 · Cited by 11
Circulating miRNome of Trachemys scripta after elective gonadectomy under general anesthesia
Bardi, E., Brizzola, S., Ravasio, G., et al. (2021) · Scientific Reports · doi:10.1038/s41598-021-94113-8 · Cited by 11
Baki, M. A. A., Jung, J. K., & Kim, Y. (2021) · Frontiers in Physiology · doi:10.3389/fphys.2021.701616 · Cited by 11
Berruien, N. N. A., Murray, J. F., & Smith, C. L. (2021) · Gene · doi:10.1016/j.gene.2021.145855 · Cited by 11
Christesen, D., Yang, Y. T., Chen, W., Batterham, P., & Perry, T. (2021) · Genetics · doi:10.1093/genetics/iyab112 · Cited by 11
Chitin deacetylase 2 is essential for molting and survival of Tetranychus urticae
Song, L., Chen, Y., An, X., Ding, C., & Bu, C. (2021) · Pesticide Biochemistry and Physiology · doi:10.1016/j.pestbp.2021.104962 · Cited by 11
Amiri, R., Tabandeh, M. R., & Hosseini, S. A. (2021) · Arquivos Brasileiros de Cardiologia · doi:10.36660/abc.20200044 · Cited by 11
Sahu, R., Vishnuraj, M. R., Srinivas, C., et al. (2021) · Journal of Microbiological Methods · doi:10.1016/j.mimet.2021.106318 · Cited by 11
Parameters to identify good quality oocytes and embryos in cattle
Wrenzycki, C. (2021) · Reproduction Fertility and Development · doi:10.1071/rd21283 · Cited by 11
Mardones, M. L., Thatje, S., Fenberg, P. B., & Hauton, C. (2021) · Marine Biology · doi:10.1007/s00227-021-03990-0 · Cited by 13
“Standard curves of 10fold serial dilutions were conducted to estimate the ampli cation e ciency and the linearity (r2), according to the MIQE guidelines (Bustin et al., 2009) with e ciency ranging between 90 – 105% and linearity greater than r2=0.”
MethodGuo, Y., Zhang, Q., Hu, X., et al. (2021) · Frontiers in Genetics · doi:10.3389/fgene.2021.795669 · Cited by 11
Cerca, N., Lima, Â., & França, Â. (2022) · Journal of Microbiological Methods · doi:10.1016/j.mimet.2022.106421 · Cited by 10
Ortega, M. A., Chaowen, C., Fraile‐Μartinez, O., et al. (2022) · Journal of Personalized Medicine · doi:10.3390/jpm12020277 · Cited by 11
“RNA was extracted following the guanidinium thiocyanate-phenol-chloroform method [27,28] allowing the analysis of mRNA expression levels of the selected genes.”
MethodPłaczkiewicz, J., Adamczyk-Popławska, M., Kozłowska, E., et al. (2022) · Pathogens · doi:10.3390/pathogens11040394 · Cited by 11
“Real-time PCR experiments were performed according to the Minimum Information for Publication of Quantitative Real-Time PCR Experiments guidelines [51].”
MethodGM-CSF Protects Macrophages from DNA Damage by Inducing Differentiation
Vico, T., Youssif, C., Zare, F., Comalada, M., Sebastián, C., Lloberas, J., & Celada, A. (2022) · Cells · doi:10.3390/cells11060935 · Cited by 12
“Data were analyzed by the ∆∆Ct method [22] using Biogazelle Qbase+ software (Biogazelle, Gent, Belgium).”
MethodBonanni, A., d’Aiello, A., Pedicino, D., Sario, M. D., Vinci, R., Ponzo, M., Ciampi, P., Curto, D. L., Conte, C., Cribari, F., et al. (2022) · Journal of Clinical Medicine · doi:10.3390/jcm11061711 · Cited by 13
“In accordance with the MIQE guidelines [15], there was full transparency on the performance of every PrimePCR assay in the form of a standardized specification and validation sheet that can be found on the Bio-Rad website, www.”
Methodde Oliveira, A. L. G., Chaves, A. T., Cardoso, M. S., et al. (2022) · Microbes and Infection · doi:10.1016/j.micinf.2022.104981 · Cited by 11
Zhang, J., Huang, Y., Xue, P., et al. (2022) · International Journal of Infectious Diseases · doi:10.1016/j.ijid.2022.04.040 · Cited by 11
Diagnostics of banana blood disease
Rincón‐Flórez, V. A., Ray, J. D., Carvalhais, L. C., et al. (2022) · Universiti Putra Malaysia Institutional Repository (Universiti Putra Malaysia) · Cited by 11
Jin, Y. J., Kong, H. G., Yang, S. I., et al. (2022) · PhytoFrontiers™ · doi:10.1094/phytofr-09-21-0069-r · Cited by 11
Fan, H., He, Q., Dong, Y., et al. (2022) · Scientific Reports · doi:10.1038/s41598-022-12782-5 · Cited by 11
Li, T., Li, H., Wu, Y., et al. (2022) · Frontiers in Immunology · doi:10.3389/fimmu.2022.905628 · Cited by 11
Filippenkov, I. B., Remizova, J. A., Denisova, A. E., Stavchansky, V. V., Golovina, K. D., Gubsky, L. V., Limborska, S. A., & Dergunova, L. V. (2022) · International Journal of Molecular Sciences · doi:10.3390/ijms23137308 · Cited by 13
“Two reference genes Gapdh and Rpl3 were used to normalize the RT–PCR results [54].”
MethodHrovat, K., Likozar, A. R., Zupan, J., & Šebeštjen, M. (2022) · Journal of Cardiovascular Development and Disease · doi:10.3390/jcdd9070211 · Cited by 13
“Gene expression measurements were performed according to the MIQE guidelines [21].”
MethodMcAlpine, S. M., Roberts, S. E., Hargreaves, B. K. V., Bullock, C., RAMSEY, S. E., Stringer, E., Lang, B. A., Huber, A. M., György, B., Erdélyi, F., et al. (2022) · The Journal of Rheumatology · doi:10.3899/jrheum.220160 · Cited by 10
Osman, F., Dang, T., Bodaghi, S., et al. (2022) · PhytoFrontiers™ · doi:10.1094/phytofr-04-22-0046-fi · Cited by 11
Honrado, M., Lopes, A. R., Pinto, M. A., et al. (2022) · Food Research International · doi:10.1016/j.foodres.2022.111761 · Cited by 11
Zhao, L., Zhang, D., Liu, Y., et al. (2022) · Foods · doi:10.3390/foods11152282 · Cited by 11
“The DNAbased sensitivity was tested by five tenfold serial dilutions from three different starting DNA concentrations (10, 5, and 2 ng/μL) [26], while the cell density-based sensitivity was measured by extracting nucleic acid from cultures of different densities (108–102 cfu/mL).”
MethodGM2 gangliosidosis AB variant: first case of late onset and review of the literature
Ganne, B., Dauriat, B., Richard, L., et al. (2022) · Neurological Sciences · doi:10.1007/s10072-022-06270-x · Cited by 11
“GM2A gene expression (Qn) was determined using the ΔCt method and the patient/control ratio by ΔΔCt method [16, 17].”
MethodBorski, A., Eskandary, F. A., Haindl, S., et al. (2022) · Transplantation · doi:10.1097/tp.0000000000004285 · Cited by 11
“for Publication of Quantitative Real-Time PCR experiments guidelines.(29) Outliers were excluded from calculations when the coefficient of variation was >1%.”
MethodLAMP for in-field quantitative assessments of airborne grapevine downy mildew inoculum
Douillet, A., Laurent, B., Beslay, J., et al. (2022) · Journal of Applied Microbiology · doi:10.1111/jam.15762 · Cited by 11
“…amplicon with the ddPCR primers and probe (the probe and primer sequences are summarized in Table S2 and Figure S1), with a slight shift in the ITS2 sequence to optimize the amplification reaction and Taq Man probe detection (Bustin et al., 2009; Raymaekers et al., 2009; Rodríguez et al., 2015).”
MethodLaboratory Plasticware Induces Expression of a Bacterial Virulence Factor
Hansen, P., von Bargen, K., Jünger‐Leif, A., & Haas, A. (2022) · mSphere · doi:10.1128/msphere.00311-22 · Cited by 11
“Assay specificity was verified by running a melt curve (47).”
BackgroundZhao, J., Yang, J., Wang, X., et al. (2022) · Genes · doi:10.3390/genes13081445 · Cited by 11
“Therefore, identifying ideal reference genes for a particular species and understanding their performance under different conditions are critical for delivering reliable gene expressional analytical results [13,24].”
BackgroundeIF3 Interacts with Selenoprotein mRNAs
Hayek, H., Eriani, G., & Allmang, C. (2022) · Biomolecules · doi:10.3390/biom12091268 · Cited by 12
“qRT-PCR reactions were performed and designed according the MIQE guidelines [44], the specificity of the oligonucleotides was validated, and the amplification efficiencies of the primer sets are all between 90 and 110% and r2 values above 0.”
MethodSarathkumara, Y. D., Browne, D. J., Kelly, A. M., et al. (2022) · International Journal of Molecular Sciences · doi:10.3390/ijms231810609 · Cited by 11
“Primer efficiencies were calculated as per MIQE guidelines [43] as previously published from cDNA standards [6].”
MethodPh5GT silencing alters flower color and flavonoids metabolome profile in petunia
Li, X., Cao, J., Zhao, H., et al. (2022) · Physiologia Plantarum · doi:10.1111/ppl.13795 · Cited by 11
Talibova, G., Bilmez, Y., & Öztürk, S. (2022) · Histochemistry and Cell Biology · doi:10.1007/s00418-022-02157-2 · Cited by 10
“Rad51, Rpa70, Ku80, and Xrcc4 gene expression analysis in the postnatal testes was carried out by qRT-PCR as in previous studies (Bustin et al. 2009; Ozturk et al. 2012, 2014; Kosebent and Ozturk 2021a).”
MethodMcMillin, S. L., Evans, P. L., Taylor, W. M., Weyrauch, L. A., Sermersheim, T. J., Welc, S. S., Heitmeier, M. R., Hresko, R. C., Hruz, P. W., Koumanov, F., et al. (2022) · Biomolecules · doi:10.3390/biom12121734 · Cited by 11
“RT-qPCR analyses were performed using methods standardized according to the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines [20].”
MethodSkibińska, I., Andrusiewicz, M., Jendraszak, M., Żbikowska, A., Jędrzejczak, P., & Kotwicka, M. (2022) · Human Cell · doi:10.1007/s13577-022-00847-6 · Cited by 11
“The quantitative PCR reactions were designed in line with the MIQE guidelines [63] (Online Resource 1, MIQE checklist and data set).”
MethodPeixoto, J. C., Saelao, P., Johnson, W. C., et al. (2022) · Frontiers in Cellular and Infection Microbiology · doi:10.3389/fcimb.2022.1093338 · Cited by 11
“CFX Manager Software (Bio-Rad) was used to obtain quantification cycle (Cq) values (Bustin et al., 2009) and analyze expression data.”
MethodUchiyama, A., Naritomi, Y., Hashimoto, Y., et al. (2022) · Bioanalysis · doi:10.4155/bio-2022-0190 · Cited by 11
Metadata
- Authors
- Bustin, S. A., Benes, V., Garson, J. A., Hellemans, J., Huggett, J., Kubista, M., Mueller, R., Nolan, T., Pfaffl, M. W., Shipley, G. L., Vandesompele, J., & Wittwer, C. T.
- Affiliations
- Queen Mary University of London (GB)
- Genomics Core Facility, EMBL Heidelberg, Heidelberg, Germany
- University College London Hospitals NHS Foundation Trust (GB)
- Ghent University Hospital (BE)
- University College London (GB)
- Czech Academy of Sciences, Institute of Biotechnology (CZ)
- Sequenom (United States) (US)
- Sigma–Aldrich, Haverhill, UK
- Technical University of Munich (DE)
- The University of Texas Health Science Center at Houston (US)
- University of Utah (US)
- Corresponding author
- Bustin, Stephen A
- ORCID
- Venue
- Clinical Chemistry
- Publisher
- Oxford University Press (OUP)
- Published
- 2009-04-01
- Volume
- 55
- Issue
- 4
- Pages
- 611-622
- ISSN
- 0009-9147 (ISSN-L)
- 1530-8561
- OpenAlex
- W2168420558
- Cited by
- 16,303
- References
- 92
- Citations by year
- 2026: 1198 · 2025: 1087 · 2024: 1107 · 2023: 1118 · 2022: 1157 · 2021: 1339 · 2020: 1264 · 2019: 1139 · 2018: 1082 · 2017: 1067 · 2016: 991 · 2015: 944 · 2014: 832 · 2013: 704 · 2012: 556
- FWCI
- 168.11
- Type
- journal article
- Language
- en
- Open access
- bronze
- doi.org
- Licence
- academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model (vor)
- Locations
- Clinical Chemistry (publishedVersion)
- PubMed (publishedVersion)
- Ghent University Academic Bibliography (Ghent University) (submittedVersion)
- UCL Discovery (University College London) (submittedVersion)
- mediaTUM – the media and publications repository of the Technical University Munich (Technical University Munich) (submittedVersion, other-oa)
- Funders
- Bowel and Cancer Research (1119105)
- ARUP Institute for Clinical and Experimental Pathology
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Molecular Biology Techniques and Applications
- Social Sciences › Decision Sciences › Statistics, Probability and Uncertainty › Meta-analysis and systematic reviews
- Social Sciences › Library and Information Sciences › Academic Writing and Publishing
- Keywords
- MIQE guidelines, quantitative real-time PCR, reporting guidelines, reproducibility, scientific integrity
- MeSH terms
- Humans, Terminology as Topic, Nucleic Acids, Publishing, Time Factors, Polymerase Chain Reaction, Molecular Diagnostic Techniques, Reverse Transcription
About these checks
Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.
A language model drew the claims from the paper on 2026-10-07, under version 3 of the claim definition (model deepseek-v4.1-flash, prompt extract-v3).
A second model labelled each claim on 2026-10-07 (model jev-1.13-20260917, definition revision gold-guideline-v0.2).
Cited sources are checked with a language model’s help (gpt-6-sol).
Statistics and arithmetic are recomputed, not judged.
Confidence bands are shown only for fields calibrated on our annotated test set. None is calibrated yet, so no band is shown.
What counts as a claim ›How we measure confidence ›How we check this ›
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