When performing qPCR on blood RNA, there is always gDNA contamination. Are there any kits with UNG for contamination prevention?

For probe-based kits with UNG, you may choose mainstream domestic brands, but I recommend selecting a probe-based master mix optimized for low-template input and containing dUTP/UNG, rather than a cheap basic version. A 2 μl RNA sample volume is not large; prepare the master mix before dispensing to avoid repeated pipetting. Sensitivity should be validated in-house: use low-copy plasmids or diluted positive cDNA for 10-fold serial dilutions, and check Ct SD and linearity. After receipt, note that UNG pretreatment at 50°C should not be extended too long, otherwise it will impair the signal of U-containing products.

DNase treatment can indeed cause RNA loss, but if gDNA is not removed, both quantification and NTC will be affected. When the RNA sample volume is small, such as 2 μl, on-column DNase treatment is recommended: digest on the column for 10–15 minutes before elution, which causes less loss than in-solution treatment. After treatment, concentration measurement will be affected by fragments and salts, so do not rely solely on concentration; an NRT can be performed to confirm gDNA removal. In practice, do not use excessive DNase, do not prolong digestion time, and after treatment, heat inactivation and brief centrifugation can be performed.

First, separate the two issues: UNG prevents aerosolized amplicon contamination, not gDNA contamination. If your NTC shows a peak, first run a melt curve or gel electrophoresis. If it matches the positive product, it may be aerosol contamination, and UNG can help; if the fragment size is consistent with gDNA amplification, you must use DNase or intron-spanning primers. After whole blood RNA extraction, it is recommended to include an NRT (no-reverse-transcriptase control). If the NRT shows a peak, it indicates gDNA carryover; otherwise, it is more likely amplicon contamination or primer dimers.

Pitfalls: When extracting RNA from whole blood, if using a column-based kit, it is recommended to perform on-column DNase I digestion before elution to avoid RNA loss from an additional separate purification step. When loading 2 μl RNA, use low-retention tips; after loading, flick gently to mix—do not vortex vigorously. After adding UNG-containing reagents, do not leave at room temperature for too long; proceed to the instrument as soon as possible according to the instructions, otherwise UNG may prematurely digest downstream dUTP-containing products. If NTC occasionally shows amplification, also check whether the primers and probes form dimers or nonspecific products.

When selecting, look at three points: whether it contains dUTP/UNG, whether it is suitable for 2 μl template, and whether it has undergone rapid testing. After arrival, first run 4-6 replicates each of NTC and NRT. NTC peak emergence should be significantly reduced, but if gDNA is still present, you must go back and optimize the DNase step. Zoned operation remains the bottom line: keep the sample addition area and amplification area separate, and always keep nucleic acid decontamination reagents on hand. I will not name specific brands, but you can look for the specification "probe-based qPCR master mix, containing UNG" and require the supplier to provide validation data for low-template blood RNA.