Minimum Information Necessary for Quantitative Real-Time PCR Experiments

The MIQE (minimum information for the publication of quantitative real-time PCR) guidelines reflect a significant milestone for the transition of a robust “gold standard” in the process of turning the accurate quantitative polymerase chain reaction (qPCR) from a testing technique into real-time quantitative polymerase reaction (QPCR). It has allowed legacy PCRs to easily be transferred to a varied methodology that provides multiple outcomes in a minimum of time for several applications. TData acquisition simplification has given the impression of stable and accurate qPCR data but fails to focus on researchers that a good qpcR assay requires several crucial steps, with each one quality managed to ensure findings are meaningful. Sadly, it has for some time been clear that the volume of qPCR data is unprecedented. As a result , multiple articles record contradictory evidence and sometimes the findings can not be replicated, but are published in the literature checked by pairs without any clear criterion to differentiate between a legitimate finding and a scientific artifact.

Guidelines:

The MIQE recommendations include a framework for reproducibility and quality assurance that allows scientists in quantitative PCR research to promote best practices. Their primary goal is to promote consistent and thorough publishing of scientific knowledge as this allows readers to accept technological competence as their own and to focus on the biological significance of the results of the report. It is a consequence that all the knowledge needed for the design, testing and optimization of a test from scratch is used as a template for successful test design. Anyone who uses MIQE to produce an assay using qPCR is practically guaranteed to achieve this objective and to achieve an accurate , precise and responsive test.

MIQE consists of nine parts with an total of 85 parameters to include the minimal details required for a possible replication, and an unequivocal estimation of the efficiency of a qPCR experiment. The following are nine sections:

  1. Experimental design
  2. Sample properties
  3. Nucleic acid extraction and quality assessment
  4. Reverse transcription
  5. Target information
  6. Primer and probe details
  7. qPCR protocol optimization
  8. qPCR validation details
  9. Data analysis

The 85 parameters fall into two categories: some are deemed to be essential and are labeled “E” in the published guidelines, because they are indispensable for an adequate description of the qPCR assay. Other components are more peripheral and are labeled “D” (desirable), yet represent an effective foundation for the implementation of best practice protocols. Adherence to these parameters also encourages much-needed standardization, especially important when using qPCR assays for diagnostic applications.

PART I: EXPERIMENTAL DESIGN

  • Definition of experimental and control groups (Essential) – Define your experimental and control groups and how many you have in each group.
  • Was the assay carried out by a core lab or the investigator’s lab? (Desirable)
  • Acknowledgement of authors’ contributions (Desirable)

PART II: SAMPLE INFORMATION

  • Description (Essential) – Where did the samples come from?
  • Volume/mass of sample processed (Desirable)
  • How is the sample collected? (Essential) – o ie. Microdissection, macrodissection, or is it simply a mass of tissue?
  • Processing procedure (Essential)
    • If frozen – how and how quickly? (Essential) – Important for good RNA yield.
    • If fixed – with what, how quickly? (Essential) –
    • Sample storage conditions and duration (especially FFPE samples) (Essential) – generally FFPE samples that are over 10 years old can be a problem.

PART III: NUCLEIC ACID EXTRACTION – a general statement is needed not for each sample.

  • Procedure and/or instrumentation (Essential) – Did you use a Polytron or bead beater to homogenize tissues or complex samples? This may NOT be required for tissue culture samples. Name of kit and details of any modifications (Essential) – Describe the kit/method used for nucleic acid extraction including the catalog number (if applicable) and any modifications from the manufacturer’s/published protocol.
  • Source of additional reagents used (Desirable) – include catalogue numbers.
  • Details of DNase or RNAse treatment (Essential) – Remember that DNase treatment is usually NOT effective for short PCR amplicon’s (ie. under 150 base pairs). Brine shrimp DNase or E. coli DNase I or II are good choices.
  • Contamination assessment (DNA or RNA) (Essential)
    • The best DNA contamination assessment for RNA is the (–) Reverse Transcriptase control.
    • DNA contamination is often overlooked especially when the primers cross exon junctions. Every assay can be different (ie. there may be a pseudo-gene).
  • Nucleic acid quantification (Essential)
    • Instrument and method for Nucleic Acid quantification (Essential) – did you use a spectrophotometer, Nanodrop, or other instrument?
    • Nucleic Acid Purity and Yield (Desirable) – Generally only a global statement made about all your samples is required.
      • RNA(A260/A230) – 1.7 or above. May not always be above 1.7 but may be okay.
      •  DNA(A260/A280) – Between 1.8-2.2 . Reviewers generally want this ratio >1.8.
  • RNA integrity: Instrument and method (Essential) – only a global statement is required. (ie The worst sample had an RIN value of 8.0.)
    • Instrument: (ie. Agilent 2100, Biorad Experion)
    • Method – RIN/RQI or Cq of 3′ and 5′ transcripts
      • RIN/RQI – Remember that RIN/RQI values DON’T necessarily correlate with mRNA quality. But it is the best we have to date.
      • Cq (Quantitation Cycle) of 3′ and 5′ transcripts – Ratio of two assays, same target one on the 3’ end and one on the 5’ end then report a ratio of the two transcripts. Ideally, the ratio should be one, but often times they are not.
  • Electrophoresis traces (Desirable) –
  • Inhibition testing (Essential) – (ie. Cq dilutions, spike or other)
  • The amplification curve shape can give an idea regarding PCR inhibition. Remember, you can have inhibition in the reverse transcription phase or the PCR reaction itself.
  • Describe the control Generally a known pure RNA sample on each plate should be used as a comparator for reverse transcription as well as PCR inhibition in the unknowns.

PART IV: REVERSE TRANSCRIPTION

  • Complete reaction conditions (Essential) – if using a kit, include the manufacturer catalog number, the gene specific primer sequence (reverse primer for PCR)
  • Amount of RNA and reaction volume (Essential)
  • Priming oligonucleotide (if using GSP) and concentration (Essential) – are you using gene specific primers, or oligo dT and/or random hexamers
  • Reverse transcriptase and concentration (Essential) – include minus reverse transcriptase control information for DNA contamination control
  • Temperature and time (Essential) – how long did this reaction run for? Some reviewers demand this information.
  • Manufacturer of reagents and catalogue numbers (Essential) – is a master mix you made yourself? If so, recipe of the master mix.
  • Cqs with and without RT (Desirable)* – is the reverse transcription enzyme a MMLV or AMV? And how many units using per reaction?
  • Storage conditions of cDNA (Desirable) -dilution factor for cDNA prior to adding to the PCR reaction. A lot of people add water to their cDNA reaction. Then they add a small volume of that to their PCR reaction. These details are considered important by some reviewers.

PART V: qPCR TARGET INFORMATION

  • What is the gene symbol? (Essential)
  • If multiplexed, efficiency and LOD (Limit of Detection) of each assay. (Essential)
  • Sequence accession number (Essential) ie. NM_########
  • Location of amplicon (Desirable)
  • Amplicon length (Essential)
  • In silico specificity screen (BLAST, etc) (Essential) – use BLAST N (looking for single hit) if you get a large hit of homology, you may want to reconsider whether you want to use that assay.
  • Pseudogenes, retropseudogenes or other homologs? (Desirable) sometimes we don’t know.
  • Sequence alignment (Desirable)
  • Secondary structure analysis of amplicon (Desirable) – M fold analysis: gives an idea of what the secondary structure of your RNA/DNA is. This will influence the efficiency of your assay.
  • Location of each primer by exon or intron (if applicable) (Essential)
    •  does it cross an intron/exon boundary?
    • are there specific splice variants that may be targeted?
  •  What splice variants are targeted? (Essential)

PART VI: qPCR OLIGONUCLEOTIDES

  • Primer sequences (Essential) – if using a predesigned assay provide the manufacturer and the catalog number.
  • RTPrimerDB Identification Number (Desirable) – if the primer and/or probe sequences are known, provide them
  • Probe sequences (Desirable)**
  • Location and identity of any modifications (Essential) – examples include Inosine, superbases, isobases MGB etc.
  • Manufacturer of oligonucleotides (Desirable)
  • Purification method (Desirable)

PART VII: qPCR PROTOCOL

  • Complete reaction conditions (Essential)
  • Reaction volume and amount of cDNA/DNA (Essential) – If it’s a kit provide the manufacturing catalog number.
  • Primer, (probe), Mg++ and dNTP concentrations (Essential)
  • Polymerase identity and concentration (Essential)
  • Buffer/kit identity and manufacturer (Essential)
  • Exact chemical constitution of the buffer (Desirable)
  • Additives (SYBR Green I, DMSO, etc.) (Essential)
  • Manufacturer of plates/tubes and catalog number (Desirable)
  • Complete thermocycling parameters (Essential)
  • Reaction setup (manual/robotic) (Desirable)
  • Manufacturer of qPCR instrument (Essential)

PART VIII: qPCR VALIDATION

  • Evidence of optimization (from gradients) (Desirable)
  • Specificity (gel, sequence, melt, or restriction digest) (Essential)… if you’re running SYBR green
  • Cq of the No Transcript Control (Essential)… for SYBR Green
  • Standard curves with slope and y-intercept (Essential)
  • PCR efficiency calculated from slope (Essential)
  • Confidence interval for PCR efficiency or standard error (Desirable)
  • R2 of standard curve (Essential)
  • Linear dynamic range (Essential)
  • Cq variation at lower limit (Essential)
  • Confidence intervals throughout range (Desirable)
  • Evidence for limit of detection (Essential)
  • If multiplex, efficiency and Limit of Detection (LOD) of each assay (Essential)

PART IX: DATA ANALYSIS

  • qPCR analysis program (source, version) (Essential) ie qBasePlus; GenEx; REST; DataAssist
  • Cq method determination (Essential) -threshold method (common) vs. 2nd derivative method
  • Outlier identification and disposition (Essential)
    • How do you figure out outliers?
    • What are the results of your no template controls?
    • Was there a value?
    • And at what cycle
  • Results of No Template Controls (Essential)
  • Justification of number and choice of reference genes (Essential) – geNorm or Norm Finder?
  • Description of normalization method (Essential) – transcripts, multiple transcripts or Ribogreen
  • Number and concordance of biological replicates (Desirable)
  • Number & stage (RT and qPCR) of technical replicates (Essential)
  • Repeatability (intra-assay variation) (Essential)
  • Reproducibility (inter-assay variation, %CV) (Desirable)
  • Power analysis (Desirable)
  • Statistical methods for result significance (Essential)
  • Software (source, version) (Essential) – what software use and how determine outliers.
  • Cq or raw data submission using RDML (Desirable)

It should be emphasized that MIQE recommends minimal guidelines; thus, if needed, more details can be released. For eg, MIQE demands precision detail, PCR effectiveness, R2 of calibration curves, linear dynamic range and limit change of CQ. Such conditions can be fulfilled by including data in a table. But it will be much more useful for the reader to include the individual scaling and melting curves in additional content to provide a much better feel for the consistency of the published details.

Another example of the comprehensive monitoring recommendations made under the MIQE Guidelines is the quality management of nucleic acids. Although the value of assessing RNA purity prior to quantification is well known to most researchers, many struggle to guarantee sufficient sample pureness. Purity is not the A260 / A280 ratio for the sample, but rather includes the absence of RT or PCR reaction inhibitors. Inhibition is a well-known yet poorly described phenomenon. The methodology called SPUD contrasts the sample prepared Cqs resulting from amplification of the water-suspended SPUD templates to those resulting from SPUD templates.

Inhibition of qPCR assays using SPUD as a reporter (a) FFPE extracted RNA samples; (b) EDTA; (c) Phenol; (d) Ethanol

Reference:

  • Quantative Real-Time PCR Methods and Protocols. Humana Press
  • MIQE Guidelines (Simplified) Soure: http://www.cenovance.com/images/MIQE.pdf

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