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The PCR amplification efficiency is low, and the bands are weak or no amplification occurs.: ① The reason for using template primers: Ensure the quality of the template and primers, and guarantee that they are not degraded. Additionally, if the template contains a large amount of inhibitors (such as plant genomic template), it is recommended to use PCR enzymes with strong inhibitor resistance; or perform gradient dilution of the template before amplification to explore the appropriate template dosage.

② Reasons for the reaction system and conditions: Increasing the number of cycles, reducing the annealing temperature, and appropriately increasing the working concentration of Mg2+ can all improve the amplification efficiency, but at the same time, it will lead to a decrease in specificity and fidelity. It is necessary to optimize the appropriate conditions according to the specific experimental requirements.

The PCR amplification has poor specificity, resulting in phenomena such as primer dimers, bands, and tailing.: ① Select heat-labile enzyme: Compared with ordinary PCR enzymes, heat-labile enzymes have higher amplification efficiency and specificity.

② Optimize reaction system and conditions: Increasing the annealing temperature can effectively enhance specificity. Additionally, reducing the number of cycles and appropriately lowering the working concentration of Mg2+ can also improve specificity, but all these measures will lead to a decrease in amplification efficiency. It is necessary to optimize suitable conditions according to specific experimental requirements.

The sample for electrophoresis was a set of oligonucleotides, but the banding positions were incorrect. Why was this the case?: The oligonucleotides should be detected on a polyacrylamide gel containing 7M urea, and should be loaded together with a 50% formamide solution to avoid compression and the formation of secondary structures. Oligonucleotides of the same length but with different compositions can produce different electrophoretic results. dC migrates the fastest, followed by dA and dT, and the slowest is dG. Oligonucleotides containing ribonucleic acid are prone to produce blurry electrophoretic bands and usually have problems with secondary structures.

When conducting direct PCR on the blood samples, the positive control showed a specific band, while the test samples showed no band or a weak band.: If the sample lysis mixture is improperly stored or stored for too long, the DNA genome has degraded. The lysis mixture can be stored at 2-8℃ for 5 days. It is recommended to use freshly prepared lysis mixture for PCR.

If the yield of reverse transcription cDNA is low or no product is produced, how should one troubleshoot this issue?: The following steps are recommended for troubleshooting:
RNA template quality: Check if the RNA is degraded; ensure that the template dosage is within the recommended range (10 pg - 2 μg).
Template type: Confirm that the total RNA or enriched miRNA is being used, rather than RNA samples that have already had small RNAs removed.
Reaction mixture: Confirm that all components are correctly added, and that the key enzyme components are kept on ice during the operation.
Reaction procedure: Confirm that the incubation temperature and time are correct.

How to select reverse transcription primers?: There are two types of primers commonly used: Anchored Oligo(dT)₂₃VN and Random Primers. You can choose them flexibly according to your experimental needs:

Oligo(dT) primers: Specifically bind to the Poly(A) tail of mRNA, suitable for reverse transcription of the vast majority of eukaryotic mRNAs, especially for full-length cDNA synthesis.

Random primers: Can bind to multiple sites on the RNA template, suitable for reverse transcription of RNA without a Poly(A) tail (such as prokaryotic RNA, rRNA, etc.) and the 5' end region of mRNA.

Mixed use: Using both types of primers together can combine their advantages, improving coverage and cDNA yield.

Why did the reverse transcription without RT control (No-RT Control) show amplification signals?: Possible causes include:

Genomic DNA contamination: Genomic DNA remaining from the RNA extraction process was amplified by PCR. It is recommended to pre-treat the RNA sample with DNase I or use reverse transcription kits with gDNA removal function.

Dimer formation of primers or non-specific amplification: Check if the qPCR primer design is reasonable. If necessary, redesign the primers.

Reagent contamination: Replace with new RNase-free water and repeat the experiment with primers. Prepare the reaction system in a laminar flow hood to reduce aerosol contamination.

Can the reverse transcription products be preserved for a long time?: The synthesized cDNA should be stored at -20℃ for short-term (1-2 weeks) and at -80℃ for long-term storage. Avoid repeated freezing and thawing, and aliquot the samples for storage to reduce the risk of degradation.

Abnormal qPCR amplification curve (plateau phase decline, poor repeatability of the wells): Possible causes and solutions:

Excessive cDNA template concentration: It is recommended to dilute the cDNA appropriately (5-10 times) before performing qPCR.

Inappropriate primer concentration: It is suggested that the final concentration of the miRNA-specific forward primer should be approximately 200 nM.

Inconsistent reaction system preparation: It is recommended to use pre-mixed solutions to reduce sampling errors. After adding the reagents, mix well and briefly centrifuge.

The qPCR melting curve shows double peaks or a broad peak.: Possible causes and solutions:

Non-specific amplification: Try raising the extension temperature from 60℃ to 65℃.

Primer dimer formation: Appropriately reduce the primer concentration and pay attention to the concentration ratio of the upstream and downstream primers.

Inappropriate primer design: It is recommended to redesign the specific forward primer to ensure its ability to specifically recognize the target miRNA sequence.

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