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| Product Name | miRNA-Specific Reverse Transcription Primer Set |
| Catalog No. | PRIPRO-0023 |
| Description | A custom-designed set of reverse transcription primers specific for microRNA (miRNA) targets. This product utilizes a stem-loop RT primer design strategy in which each primer contains a miRNA-specific 3′ overhang (typically 6–8 nucleotides complementary to the 3′ end of the target miRNA) and a universal stem-loop sequence that serves as a template for subsequent qPCR amplification. The stem-loop structure increases primer thermal stability and prevents priming of precursor miRNA (pri-miRNA and pre-miRNA), ensuring that only mature miRNA species are detected. This design enables detection of miRNAs as short as 18–25 nucleotides with high sensitivity and specificity, even discriminating between closely related miRNA family members differing by a single nucleotide (e.g., let-7 family). Custom primer sets are designed for single-plex or multiplex reverse transcription to detect panels of miRNAs from limited RNA samples. |
| Intended Use | Designed for specific and sensitive detection of mature microRNAs by stem-loop RT-qPCR in applications including miRNA expression profiling, circulating miRNA biomarker discovery, functional miRNA studies, and validation of miRNA-seq results. |
| Principle / Technology | Stem-loop RT primer with 3′ miRNA-specific extension (6–8 nt) and universal stem-loop backbone; reverse transcriptase extends only when miRNA is perfectly paired at 3′ end; stem-loop structure increases specificity and prevents pre-miRNA priming |
| Detection Method | Stem-loop reverse transcription followed by miRNA-specific qPCR with TaqMan probe or SYBR Green detection; signal proportional to mature miRNA copy number |
| Sample Type | Total RNA (including small RNA fraction) from cells, tissues, serum, plasma, urine, exosomes, and FFPE specimens; 1 ng – 1 µg total RNA per RT reaction |
| Performance Range / Specifications | Detects mature miRNAs of 18–25 nt; loop length optimized for target miRNA; discriminates miRNAs with single-nucleotide differences; linear detection range 6–7 orders of magnitude |
| Sensitivity / LOD | Detection of fewer than 10 copies of miRNA per reaction; capable of detecting low-abundance circulating miRNAs from as little as 10 µL serum |
| Specificity | Single-nucleotide discrimination between miRNA family members; no cross-reactivity with precursor forms (pri-/pre-miRNA); stem-loop design provides higher specificity than linear RT primers for short RNA targets |
| Reaction Conditions / Protocol | RT: combine RNA with miRNA-specific stem-loop RT primer(s), dNTPs, and reverse transcriptase; pulse-spin and incubate at 16 °C for 30 min (stem-loop annealing), 42 °C for 30 min (extension), 85 °C for 5 min (inactivation). qPCR: use 1–2 µL RT product with miRNA-specific forward primer and universal reverse primer. |
| Components / Formulation | Set of stem-loop RT primers (one per target miRNA); lyophilized or in solution; universal reverse primer included; optional miRNA-specific qPCR forward primers and probes |
| Storage Conditions | –20 °C; protect from light if fluorescent probes included; minimize freeze-thaw |
| Shelf Life | 12 months at –20 °C; 6 months at 2–8 °C in solution |
| Package Specifications | Set of 1–20 miRNA-specific RT primers in individual tubes or pre-mixed panel; 5–50 pmol/µL ready-to-use |
| Product Form | Lyophilized white powder or colorless solution |
| Quality Control | HPLC purity ≥90%; MALDI-TOF MS; functional QC: RT-qPCR of synthetic miRNA standards demonstrating linearity (R² >0.99)and efficiency (90–110%); no amplification in no-template controls; no cross-reactivity with non-target miRNAs tested. |
| Key Features | Stem-loop design for mature miRNA specificity; single-nucleotide discrimination; prevents pre-miRNA priming; custom panel design service; compatible with limited RNA input; detectable from serum and plasma; includes universal reverse primer. |
| Purity | ≥90% HPLC; stem-loop structure verified by secondary structure prediction; MS mass confirmed |
| Concentration | RT primers at 5 µM working concentration (or 100 µM stock for custom dilution); concentration verified by A260 |
| Activity / Unit Definition | RT efficiency >90% for target miRNA; qPCR efficiency 90–110% across 6-log dilution series; linearity R² >0.99; no amplification from 1-nt mismatched miRNA at Ct >35 |
| Molecular Weight | Typically ~15–20 kDa per stem-loop RT primer (48–65 nt); exact mass reported on COA per sequence |
| Source / Origin | Synthetic DNA oligonucleotides; stem-loop design is fully synthetic with no biological templates |
| pH Range / Optimal pH | Stable and functional in RT buffer pH 7.5–8.5; qPCR buffer pH 8.0–8.5 |
| Shipping Conditions | Ambient temperature (lyophilized) or cold pack (solution); stable during transit |
| Expiration Date / Stability | 12 months at –20 °C dry; 6 months at –20 °C in solution; aliquot RT primers to avoid freeze-thaw degradation |
| Regulatory / Compliance | For research use only; not for diagnostic applications; manufactured under ISO 9001 |
| Compatibility | Compatible with all major reverse transcriptases (SuperScript III/IV, Maxima, PrimeScript); compatible with common qPCR master mixes; applicable to ABI, Bio-Rad, Roche, and Qiagen qPCR platforms |
| Recommended Buffer System | Nuclease-free water or TE buffer pH 8.0; compatible with standard RT and qPCR buffer systems |
| Application Notes / Precautions | For multiplex RT, combine up to 5–10 miRNA-specific RT primers in a single reaction. Use a synthetic miRNA standard curve for absolute quantification. For serum/plasma samples, spike in a non-human miRNA (e.g., cel-miR-39) as an extraction and RT control. |
| Batch-to-Batch Consistency | RT-qPCR efficiency maintained within 90–110%; Ct shift <0.5 cycles across batches for same miRNA standard; MS mass accuracy ±0.05% |
For research use only, not for clinical use.
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