Q1: How to improve the sensitivity and specificity of qPCR?

Answer:

1. First, ensure that the template RNA is intact and free from DNA contamination.

2. The RNA template should not contain any inhibitors of the amplification reaction.

3. To prevent template degradation, add the RNase inhibitor RNasin to the reaction system.

4. Use an appropriate amount of template RNA—excessive amounts may reduce specificity, while insufficient amounts may result in no amplification or weak bands.

5. If the template contains secondary structures, increasing the reverse transcription temperature may improve amplification efficiency.

6. When designing primers, avoid complementary sequences at the 3’ end and prevent the formation of internal hairpin structures.

Q2: What are the methods to prevent RNA degradation?

Answer:

1. Analyze the RNA on a denaturing gel before verifying its integrity.

2. Isolate the RNA using proper aseptic techniques to avoid contamination.

3. Immediately extract RNA from tissues after dissection, and convert the extracted RNA into cDNA for cryopreservation.

Q3: How to handle RNA samples containing reverse transcription inhibitors?

Answer:

Reverse transcription inhibitors include: SDS, EDTA, glycerol, sodium pyrophosphate, spermidine, and guanidinium salts. To detect the presence of inhibitors in RNA samples, a control RNA can be mixed with the sample and the reaction yield compared with that of the control RNA alone. If the yield decreases when the control RNA is mixed with the sample, it indicates the presence of reverse transcription inhibitors in the sample. In such cases, the RNA precipitate can be washed with 70% (v/v) ethanol to remove the inhibitors.

Q4: How to resolve insufficient primer annealing during first-strand cDNA synthesis?

Answer:

Determine an appropriate annealing temperature for the primers used in the experiment. For random hexamers, it is recommended to incubate at 25°C for 10 minutes prior to the reaction temperature incubation.

Q5: How to minimize secondary structures in RNA templates?

Answer:

1. Denature the RNA and primers under salt-free and buffer-free conditions, then anneal them; increase the reverse transcription reaction temperature.

2. When the temperature exceeds 60°C, do not use oligo(dT) primers. Instead, select a gene-specific primer (GSP) that can anneal at the reaction temperature.

3. For RT-PCR products longer than 1 kb, maintain the reaction temperature at 65°C.

Q6: How to address DNA contamination in RNA samples?

Answer:

1. If the contamination is from genomic DNA, treat the RNA with DNase I; simultaneously include a no-reverse-transcription control reaction to detect DNA contamination.

2. If the contamination is from exogenous DNA, use aerosol-resistant tips and UDG (uracil-DNA glycosylase) enzyme.

Q7: What are the general principles for qPCR probe design?

Answer:

1. The amplicon length should generally not exceed 300bp. Shorter fragments are preferred.

2. The probe must not be complementary to either primer. While maintaining specificity, keep the probe as short as possible, preferably ≤30bp.

3. The probe's Tm should be at least 5°C higher than the primers' Tm values.

4. For polymorphism detection, position the variant site near the center of the probe.

5. Avoid guanine (G) at the 5' end of the probe, as it can quench the reporter fluorescence.

Q8: Amplification was observed in the no-reverse-transcriptase control RNA.

Answer:

1. During in vitro transcription, it is impossible to completely eliminate all DNA templates, so the control group may contain trace amounts of DNA. To minimize the impact of DNA contamination, it is recommended to dilute the first-strand cDNA by 1:10, 1:100, and 1:1000.

2. The band may be from primer dimers.

Q9: Amplification products remain in the loading well.

Answer:

1. High-molecular-weight DNA smear in PCR results may be caused by excessive template input. It is recommended to dilute the first-strand cDNA 100-fold before performing a second round of amplification.

2. During the second round of PCR, if the annealing temperature used is 5°C lower than the primer Tm, consider increasing the annealing temperature or using a hot-start protocol to improve specificity.

Q10: No Ct signal detected

Answer:

1. Insufficient reaction cycle parameters. Generally, it should be set to more than 35 cycles, but excessive cycles may increase background noise.

2. Incorrect fluorescence signal detection steps. The SYBR Green method (SG method) collects fluorescence signals during the 72°C extension step, while the TaqMan method collects signals either at the end of annealing or extension.

3. Degradation of primers or probes. PAGE electrophoresis can be used to check their integrity. If the electrophoresis bands appear smeared, consider re-synthesizing the primers or probes.

4. Insufficient or degraded template. In this case, re-extract the nucleic acid template.

Q11: Delayed Ct values observed (Ct>38)

Answer:

1. Low amplification efficiency due to suboptimal reaction conditions; suggested to reduce annealing temperature and increase magnesium ion concentration.

2. Degradation of reaction components or insufficient loading volume.

3. Excessive PCR product length (recommended amplicon size: 80-150 bp).

Q12: Poor linearity in the standard curve.

Answer:

1. Pipetting errors occurred, resulting in non-gradient sample concentrations.

2. The standard is degraded; avoid repeated freeze-thaw cycles.

3. Poor design of primers or probes.

4. Presence of inhibitors in the template or excessive template concentration.

Q13: Multiple dominant peaks are present in the melting curve.

Answer:

1. The primer design is not optimal.

2. Suboptimal primer concentrations with an unbalanced ratio between forward and reverse primers.

3. Excessive magnesium ion concentration.

4. Contamination of the template genome.

Q14: In the same sample, one particular fluorescent signal is exceptionally strong.

Answer:

1. During reagent preparation, the reaction solution was not completely dissolved, resulting in an increased amount of probe in one tube.

2. During reagent preparation, insufficient mixing led to inconsistent amounts of components in different tubes.

3. The PCR instrument’s heating block was contaminated with fluorescent substances, and the contamination in the heating block needs to be cleaned.

Q15: Why is there an upward or downward spike in the amplification curve?

Answer:

1. The voltage is unstable during the reaction process.

2. Around the 20th cycle, the instrument may have stopped or the cover was opened, causing a sudden increase in light intensity.

3. If the spike is downward, it may be caused by the aging of the halogen lamp, which should be replaced at this point.

Q16: Why is the amplification efficiency too low for some samples?

Answer:

1. Residual extraction solution partially inhibited the PCR reaction.

2. The reaction solution was not strictly measured, mixed thoroughly, or aliquoted evenly.

3. The reagent has expired/degraded.

Q17: Negative control or blank control shows an upward tailing.

Answer:

1. Contamination in the template extraction environment or during handling

2. Contamination occurred during reagent/solution preparation

Q18: Linear amplification curve

Answer：

1. Partial degradation of the probe: Generally, diluted probes can be stored at 4°C for at least 3 months. Repeated freeze-thaw cycles of the probe may lead to degradation, or the probe may have been exposed to light for too long.

2. Presence of PCR inhibitors in the reaction solution.

Q19: No amplification curve detected

Answer:

1. Incorrect PCR parameter settings: The fluorescence signal reading was mistakenly programmed in the first step (Stage 1) of the reaction cycle.

2. The computer was set to automatic sleep mode.

Q20: Baseline Decline

Answer:

Incorrect baseline selection range. Try increasing the baseline range – this issue is often caused by reagent quality issues.

 Original link：https://zhuanlan.zhihu.com/p/78193892