MIQE Checklist for qPCR – Copy

Number Analysis
1. MIQE Essential Checklist
The MIQE (Minimum Information for Publication of Quantitative Real-Time PCR
Experiments) guidelines provide a framework for transparent and reproducible qPCR
experiments. Below is the essential checklist adapted for copy number analysis.

• Experimental design: Description of experimental setup, controls, and replicates.

• Sample description: Source, biological replicates, sample processing.

• Nucleic acid extraction: Method, purity, integrity assessment.

• Reverse transcription (if applicable): Primers, enzyme, reaction conditions.

• qPCR target information: Gene name, accession number, amplicon length.

• Primer and probe sequences: Provided or cited; specificity verified.

• Reference gene: Justification for stability; preferably a single-copy gene for copy number
analysis.

• qPCR protocol: Cycling conditions, reaction volumes, reagent brands.

• Instrument: Make and model.

• Calibration/standard curve: Serial dilutions, efficiency, R² value.

• No-template control (NTC) and no-amplification control (NAC).

• Data analysis: Cq determination method, baseline correction, efficiency correction.

• Statistics: Replicate analysis, error propagation.

• Compliance with MIQE reporting format.

2. Specificities for Copy Number Analysis

When performing copy number analysis by qPCR, additional considerations include:

• Reference gene must be confirmed as a true single-copy gene per haploid genome.

• Test gene and reference gene amplicon efficiencies must be comparable (90–110%).
• Use at least 5–7 points for standard curves to calculate efficiency and R².

• Run all standards and samples in technical triplicates.

• Validate absence of primer-dimers and nonspecific amplification by melt curve or gel

electrophoresis.

• Normalize copy number ratio as: 2^(-ΔΔCq) or using standard curve absolute
quantification.

• Assess reproducibility across independent biological replicates.

3. Mathematics for Data Analysis

Key formulas used in qPCR copy number analysis:

• Efficiency (E) = (10^(-1/slope) - 1) × 100%

• Relative Copy Number = 2^(-ΔΔCq), where ΔΔCq = (Cq_target_sample - Cq_ref_sample) -

(Cq_target_calibrator - Cq_ref_calibrator)

• Absolute Copy Number = 10^((Cq - intercept)/slope)

• Limit of Detection (LOD): Lowest concentration reliably distinguished from zero

(commonly mean_blank + 3×SD_blank)

• Limit of Quantification (LOQ): Lowest concentration quantified with acceptable precision

(commonly mean_blank + 10×SD_blank)

4. Assay Validation Steps

• Primer specificity verification (BLAST and melt curve).

• Standard curve generation over a wide dynamic range (≥ 5 logs).

• Efficiency calculation (acceptable range: 90–110%).

• Linearity check (R² ≥ 0.99).

• Precision assessment (intra- and inter-assay variation).

• Reproducibility check across independent runs.

• Stability check of reference gene under experimental conditions.

• Calculation of LOD and LOQ.

No — don’t re-run a full LOD/LOQ experiment on every plate.
Do determine LOD and LOQ thoroughly during assay validation, then monitor
them on every plate with small QC checks (blanks + a low-copy standard point(s)) so
you can detect drift. Below I give a practical, MIQE-compatible workflow, exact
calculations, replicate guidance, acceptance rules, and what to do if a plate fails the QC.

1) Two different things: determine vs monitor

 Determine LOD/LOQ (one-time, validation stage): a rigorous experiment to

establish the assay’s true sensitivity and quantitative limit. This requires many
replicates across low concentrations and statistical analysis.
 Monitor LOD/LOQ (every plate): small checks (blanks and low-copy
control(s)) to confirm the assay is performing consistently with the validated
LOD/LOQ. You do not repeat the full validation every plate.

2) How to determine LOD & LOQ during validation (recommended, rigorous)

(Do this once during assay validation or whenever anything about the assay changes —
new master mix, new instrument, new primer lot, etc.)

A. Blank / No-template controls (NTC):

 Run 20 blank/NTC replicates (or as many as practical) to characterize

instrument/background noise.

B. Low-concentration panels:

 Prepare a panel of low-concentration standards around the expected LOD/LOQ

(e.g., 10, 5, 2, 1, 0.5 copies/µL or appropriate units).
 Run ≥20 replicates per concentration (CLSI recommends high replicate counts for
LOD assessment).

C. Calculate empirically:

 LOD (empirical): lowest concentration with ≥95% detection probability (i.e.,

≥19/20 positives).
 LOQ (empirical): lowest concentration at which quantification is acceptably
accurate and precise — common criteria: CV ≤ 20% and mean measured
concentration within ±25% (or your lab’s acceptance) of the true concentration.

D. Blank-based approach (simple):

  Compute blank mean Cq (µ_blank) and SD_blank.
 LOD_Cq ≈ µ_blank + 3 × SD_blank.
 LOQ_Cq ≈ µ_blank + 10 × SD_blank (this is conservative; use empirical LOQ if
available).
 Convert Cq thresholds to copies/µL using the standard curve:

copies/µL=10Cq−bm\text{copies/µL} = 10^{\frac{\text{Cq} - b}{m}}

where mm = slope, bb = intercept of the standard curve.

E. Document everything — methods, #replicates, acceptance thresholds — as part of

validation records and include in your MIQE reporting.

3) What to run on every plate (monitoring QC)

Include small QC elements every plate to ensure the validated LOD/LOQ still applies:

 Blanks / NTCs: at least 1–2 NTCs in triplicate (many labs use 2 distinct NTCs in
triplicate).
 Low-copy QC standard(s): include one or two low-concentration standard
points near your LOQ (triplicate). For example, include the validated LOQ point
and one point ~2–3× LOQ.
 Inter-run calibrator (IRC): triplicate.
These checks allow you to detect upward shifts in background or loss of
sensitivity without doing a full validation.

4) Practical replicate guidance

 For routine plates: NTCs and low-copy QC in technical triplicate.

 For special monitoring (e.g., weekly/monthly): run the low-copy QC in more
replicates (6–12) to get a tighter estimate of performance.
 For re-validation or final LOD determination: use the high replicate counts
described above (≥20) per CLSI-style methods.

5) Acceptance rules / flags (example, tune to your lab)

 NTC rule: No amplification or Cq > predefined cutoff (e.g., >40) for NTCs. Any
true amplification in NTCs → investigate contamination; do not accept plate
unless contamination source explained and re-run.
  Low-copy QC rule: The low-copy standard near LOQ must be detected in ≥X%
replicates (e.g., ≥95% for LOD; for routine monitoring you may require ≥66–75%
detection to keep throughput). If detection rate or mean Cq shifts >0.3 Cq vs
expected → flag plate.
 IRC rule: IRC mean Cq must be within ±0.3 Cq (or lab-determined tolerance) of
expected. If not, fail plate and investigate.
 Action on fail: If any QC fails, investigate reagents/pipetting/plate
seal/instrument, then re-run the plate (after resolving cause).

6) Calculations you should use (concrete formulas)

 Blank mean & SD:

μblank=mean(Cq of blanks),σblank=SD(Cq of blanks)\mu_{\text{blank}} = \

text{mean(Cq of blanks)},\quad \sigma_{\text{blank}} = \text{SD(Cq of blanks)}

 LOD_Cq (blank method):

LOD_Cq=μblank+3σblank\text{LOD\_Cq} = \mu_{\text{blank}} + 3\sigma_{\

text{blank}}

 LOQ_Cq (blank method, conservative):

LOQ_Cq=μblank+10σblank\text{LOQ\_Cq} = \mu_{\text{blank}} + 10\sigma_{\

text{blank}}

 Convert to copies/µL using standard curve (m = slope, b = intercept):

copies/µL=10Cq−bm\text{copies/µL} = 10^{\frac{\text{Cq} - b}{m}}

 Empirical LOD: lowest concentration with ≥95% positive detection in many

replicates.
 Empirical LOQ: lowest concentration meeting precision (CV) and accuracy
thresholds:

CV=SD of copiesmean copies×100%\text{CV} = \frac{\text{SD of copies}}{\

text{mean copies}} \times 100\%

Require CV ≤ e.g., 20%.

7) Example workflow you can adopt immediately

1. Validation stage (once):

o Run full standard curves and LOD/LOQ experiments with high replicate
counts (as above). Record m, b, σ_m, σ_b, empirical LOD/LOQ.
2. Routine plates (every run):
o Include NTCs (2× triplicate), LOQ QC point (triplicate) and IRC
(triplicate).
o If NTC or LOQ QC or IRC fails → do not accept plate; troubleshoot & re-
run.
3. Periodic re-validation:
o Every week/month or every N plates (depending on throughput) run a full
standard curve and more robust low-conc replicates to reconfirm
LOD/LOQ.
4. When to re-validate fully: any change in primer lot, master mix, instrument, or
major SOP change.

8) Adding this to your files

I can update your Excel template to:

 Add a LOD_LOQ validation sheet with fields to paste many blank/low-conc

replicates and automatically compute empirical LOD/LOQ (95% detection),
blank-based LOD/LOQ, CVs, and flags.
 Add a routine QC sheet that takes the per-plate NTCs, low-copy QC and IRC
values and returns PASS/FAIL based on your chosen thresholds (e.g., IRC Δ ≤
0.3 Cq; NTCs undetected; LOQ QC detected ≥ 2/3).
 Add automated messages/conditional formatting that highlight flagged plates.

Would you like me to add both the validation LOD/LOQ analysis sheet and the per-
plate QC check sheet to your existing workbook now?
Perfect — let’s simplify everything into a clear, step-by-step Standard Operating
Procedure (SOP) for starting any new qPCR assay, including copy number analysis,
MIQE compliance, and practical workflow. I’ll keep it concise, actionable, and structured
so you can follow it plate by plate.

SOP for New qPCR Assay (Copy Number / Absolute Quantification)

1. Assay Design & Primer Validation

1. Design primers/probe specific to your target (GOI) and reference gene (single
copy).
2. Check in silico specificity (BLAST or similar).
3. Test primers on template-positive and template-negative controls.
4. Confirm:
o Single melting peak (SYBR Green assays)
o Single band (if gel verification)

2. Standard Curve Preparation

1. Prepare a template stock (plasmid, amplicon, or genomic DNA).

2. Make ≥5–6 serial 10-fold dilutions covering at least 3 logs, ideally 5–6 logs.
o Example: 10⁶ → 10¹ copies/µL.
3. Run each dilution in technical triplicate.
4. Include low-copy QC points near expected LOQ.

3. Plate Setup

For each new assay or plate:

 Target gene: triplicates per sample.

 Reference gene: triplicates per sample.
 Standard curve: triplicates per dilution.
 Negative controls (NTC): ≥2 in triplicate.
 Inter-run calibrator (IRC): triplicate (same sample/aliquot every plate).
 Optional: Include 1–2 low-copy points to monitor LOD/LOQ per plate.

Tip: Run target and reference on the same plate if possible. If not, use a consistent IRC to
normalize between plates.
4. qPCR Run

1. Use consistent reagents, instrument settings, and reaction volume.

2. Record raw Cq/Ct values.
3. Check amplification curves for artifacts or abnormalities.

5. Standard Curve Analysis

1. Plot Cq vs log10(template copies).

2. Fit linear regression → get slope (m), intercept (b), R², efficiency (E =
10^(-1/m)-1).
3. Determine Linear Dynamic Range (LDR):
o Highest → lowest dilution showing linear behavior (R² ≥ 0.99, stable
efficiency, residuals small).
o Exclude points outside linear range (e.g., very low detection with high
CV).
4. Record:
o LDR range (copies/µL)
o Efficiency ± SE or 95% CI
o R² of the linear portion
o CV and detection % for lowest LDR point

6. Copy Number Calculation

1. Convert sample Cq to log10(copy number) using:

log10(copies)=Cq−bm\text{log10(copies)} = \frac{Cq - b}{m}

2. Apply IRC correction if target and reference are run on different plates:

badj=b+(CqIRC,plate−CqIRC,expected)b_{\text{adj}} = b + (\text{Cq}_{IRC,plate} - \
text{Cq}_{IRC,expected})

7. LOD & LOQ

 Determined during assay validation, not every plate:

o LOD: lowest concentration detected ≥95% of replicates
o LOQ: lowest concentration with acceptable precision (CV ≤ 20–35%)
  Routine monitoring per plate:
o Triplicate low-copy QC points near LOQ
o Triplicate NTCs
o Triplicate IRC

8. Plate QC Acceptance Rules

 NTCs: No amplification (or Cq >40)

 Low-copy QC: Detection meets ≥2/3 replicates (or lab threshold)
 IRC: Mean Cq within ±0.3 cycles of expected
 Efficiency and slope within lab-established ranges
 If any fail → troubleshoot & re-run

9. Replicates

 Technical triplicates for all samples, standards, and controls.

 Optional extra replicates for validation or low-copy samples.

10. Documentation

 Save raw Cq data, plate layout, and standard curve calculations.

 Record:
o Standard curve slope, intercept, efficiency ± CI/SE
o LDR
o LOD/LOQ
o QC results
 Include residual plots or prediction intervals for LDR if publishing.

11. Periodic Validation

 Full standard curve and LOD/LOQ re-validation:

o New primer/probe lot
o New master mix
o New instrument
o Major SOP change
 Otherwise, monitor plate-to-plate using IRC and low-copy QC points.
✅ Outcome: Following this SOP ensures:

 MIQE-compliant reporting
 Reliable absolute quantification / copy number calculation
 Reproducibility across plates and runs

If you want, I can turn this SOP into a one-page visual checklist with Excel templates
for:

 Standard curve analysis (slope, intercept, efficiency, CI/SE)

 LDR identification
 IRC correction
 Plate QC flags

It would be like a ready-to-use workflow for every new assay.

Do you want me to prepare that?