qPCR Setup Protocol¶

Using Solarbio 2×SYBR Green qPCR Master Mix (SR1110) or 2×TaqMan qPCR Master Mix (SR1120). This protocol covers both two-step RT-qPCR (cDNA template) and direct DNA qPCR.

Equipment and Reagents Checklist¶

Item	Recommended Product	Notes
2×SYBR Green qPCR Master Mix	SR1110 (Solarbio)	Includes ROX; verify instrument ROX requirements
2×TaqMan qPCR Master Mix	SR1120 (Solarbio)	Includes ROX; for probe-based assays
Nuclease-free water	R1600 (Solarbio)	DEPC-treated or ultra-pure
cDNA template	PC1170-generated (Solarbio)	Diluted 1:5–1:20 in nuclease-free water
qPCR tubes/plate	0.1 or 0.2 mL thin-wall	White plates improve fluorescence detection
Optical seal	Transparent adhesive film	Ensure no bubbles over wells
Real-time PCR instrument	ABI 7500, Bio-Rad CFX96, Roche LC480, etc.	—
Pipettes (0.5–10 μL, 10–100 μL)	Calibrated ±2% accuracy	Pre-wet tips before aspirating master mix

Primer and Probe Design¶

SYBR Green Primers¶

Parameter	Recommendation	Verification Method
Amplicon length	70–200 bp	Check by gel or melt curve
Primer length	18–24 nt	In silico analysis
GC content	40–60%	OligoAnalyzer / Primer3
Tm	58–62°C	Nearest-neighbor calculation
Tm difference (pair)	≤2°C	Compare forward/reverse
3′ stability	≤3 G/C in last 5 bases	Manual check
Avoid	≥4 consecutive G residues	In silico
Avoid	Primer-primer complementarity (3′ end)	Check with AutoDimer or Primer-BLAST

TaqMan Probe¶

Parameter	Recommendation
Probe length	18–25 nt
Primary Tm	68–70°C (10°C above primers)
GC content	40–50%
Reporter dye (5′)	FAM, VIC, or HEX
Quencher (3′)	BHQ-1 (FAM, VIC) or TAMRA
Avoid	≥4 consecutive G bases (reduces fluorescence)
Avoid	5′ end must not be G (quenches reporter)
Secondary structure	Check with mFold (ΔG > −3 kcal/mol)
Concentration (final)	0.1–0.3 μM

Reaction Setup (20 μL)¶

SYBR Green¶

Component	Volume (20 μL)	Final Concentration	Notes
2× SYBR Green qPCR Master Mix	10 μL	1×	Contains polymerase, dNTPs, SYBR, ROX
Forward primer (10 μM)	0.4–0.6 μL	0.2–0.3 μM	Start at 0.3 μM; optimize 0.1–0.5 μM
Reverse primer (10 μM)	0.4–0.6 μL	0.2–0.3 μM	Keep forward:reverse ratio 1:1
Template cDNA	1–2 μL	1–100 ng	Dilute cDNA 1:5 to 1:20
Nuclease-free water	To 20 μL	—	—

Important: Prepare a master mix (all components except template) for reproducibility. Include ≥10% excess volume.

TaqMan¶

Component	Volume (20 μL)	Final Concentration
2× TaqMan qPCR Master Mix	10 μL	1×
Forward primer (10 μM)	0.4 μL	0.2 μM
Reverse primer (10 μM)	0.4 μL	0.2 μM
Probe (10 μM)	0.3 μL	0.15 μM
Template cDNA	2 μL	1–100 ng
Nuclease-free water	To 20 μL	—

ROX Adjustment (if needed)¶

Instrument Requirement	Action
High ROX (ABI 7500, QuantStudio)	Use SR1110/SR1120 as-is
No ROX (Bio-Rad CFX, Roche LC480)	Use as-is; ROX channel ignored
Passive reference not used	Disable ROX detection on instrument software

Experimental Controls¶

Control Type	Composition	Expected Result	Purpose
NTC (no-template control)	Water instead of template	No Ct or Ct > 38	Detects contamination
NRT (no-RT control)	RNA sample without RT enzyme	No Ct or Ct > 35	Detects gDNA contamination
Positive control	Known template + validated primers	Ct consistent (±0.5)	Validates assay function
Inter-run calibrator	Aliquoted reference sample	Ct SD < 0.5 across runs	Normalizes between plates

Thermal Cycling¶

Step	Temperature	Time	Cycles
Polymerase activation	95°C	2 min	1
Denaturation	95°C	10–15 s	40
Anneal/Extend (plate read)	60°C	30 s (45 s for TaqMan)	40
Melt curve (SYBR only)	65–95°C, 0.5°C step	5 s each step	1

Fast Cycling Protocol (compatible with SR1110/SR1120)¶

Step	Temperature	Time	Cycles
Polymerase activation	95°C	30 s	1
Denaturation	95°C	3–5 s	40
Anneal/Extend (read)	60°C	15–20 s	40
Total run time	—	~35 min	—

Note: Fast protocol requires a fast thermal cycler (ABI 7500 Fast, Bio-Rad CFX96).

Standard Curve Preparation¶

Protocol¶

Prepare a 10-fold dilution series of the standard (plasmid, gBlock, or validated cDNA) covering 5–7 logs
Typical dilution range: 10⁷ to 10¹ copies/μL (or 1:10 serial dilution of high-abundance cDNA)
Run each dilution in triplicate

Standard Curve Layout Example¶

Dilution	Expected Copies/Reaction	Expected Ct (100% efficiency)
10⁷	2 × 10⁷	~15
10⁶	2 × 10⁶	~18.3
10⁵	2 × 10⁵	~21.7
10⁴	2 × 10⁴	~25
10³	2 × 10³	~28.3
10²	2 × 10²	~31.7
10¹	2 × 10¹	~35
NTC	0	Undetermined

Data Analysis¶

Standard Curve Method¶

Parameter	Requirement
Efficiency	90–110% (slope −3.6 to −3.1)
R²	≥0.99
Dynamic range	≥5 logs
NTC (no-template control)	Ct > 38 or no amplification
Ct SD within replicates	≤ 0.25 at > 100 copies

ΔΔCt Relative Quantification¶

Formula	Description
ΔCt = Ct(target) − Ct(reference)	Normalization to housekeeping gene
ΔΔCt = ΔCt(sample) − ΔCt(calibrator)	Fold change relative to calibrator
Fold change = 2^(-ΔΔCt)	Assuming 100% amplification efficiency

Assumptions for ΔΔCt: - Both target and reference genes amplify with ≥ 95% efficiency - Efficiency difference between target and reference ≤ 0.1 - Reference gene expression is stable across all experimental conditions

Melting Curve Analysis (SYBR Green Only)¶

Observation	Interpretation	Action
Single peak at predicted Tm	Specific amplification	OK
Double peak (Tm difference < 2°C)	Two close amplicons	Redesign primers; increase annealing Tm
Peak at 70–75°C	Primer-dimer	Reduce primers to 0.15 μM; redesign
Broad peak	Non-specific amplification	Run gel; increase annealing temperature
Peak at Tm > 85°C	Possible gDNA amplification	Use intron-spanning primers; DNase treat

Troubleshooting¶

Issue	Cause	Solution
No amplification	Probe degraded	Re-order probe; check storage at -20°C dark
	No cDNA	Verify RT reaction success (run positive control)
	cDNA diluted too much	Reduce dilution; use neat cDNA
	Inhibitor present	Dilute cDNA 1:10 or 1:20
High Ct (>35)	Low expression target	Increase cDNA to 100 ng per 20 μL reaction
	Inefficient primers	Check efficiency; redesign if < 90%
	Poor RNA quality	Check RIN; re-extract if RIN < 5
	ROX/primer mismatch	Verify instrument ROX settings
Non-specific amplification	Primer dimer	Reduce primers to 0.1–0.2 μM; melt curve analysis
	gDNA contamination	DNase treat RNA; use intron-spanning primers
	Annealing temperature too low	Increase to 62°C; test gradient 58–65°C
Poor efficiency (< 80%)	Inhibitor in sample	Dilute cDNA 1:10; purify template
	ROX mismatch	Verify instrument ROX configuration
	Pipetting error	Calibrate pipettes; pre-wet tips; use master mix
High replicate variability	Inconsistent pipetting	Use larger volume (20 μL); pre-wet tips
	Evaporation at plate edges	Use adhesive seal; pre-warm lid to 105°C
	Uneven thermal block	Use center wells; avoid edge wells for critical samples
Late Ct in NTC	Contamination (amplicon or plasmid)	Replace water; UV work area; use fresh tips
	Primer-dimer amplified	Reduce primer concentration
Baseline drift	Background adjustment incorrect	Set baseline cycles 3–15 manually

5.1 NTC Acceptance Criteria¶

Chemistry	NTC Requirement	Action if Failed
SYBR Green	Ct > 38 or undetermined	Redesign primers; re-purify or replace reagents
TaqMan	Ct > 40 or undetermined	Check for probe degradation; check for target contamination
RT-qPCR	NRT Ct — ΔCt > 10 vs. RT+	DNase treat RNA; redesign primers

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