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| Product Name | miRNA 1st Strand cDNA Synthesis Kit (Poly(A) Tailing Method) |
| Catalog No. | RESK-0024 |
| Description | MicroRNAs are a widely studied class of non-coding RNAs of about 22 nt that play important regulatory roles in gene expression in plants and animals. This kit uses the poly(A) tailing method for first-strand cDNA synthesis from miRNA. The 2x miRNA RT buffer contains all reagents and primers required for both the miRNA poly(A) tailing reaction and the reverse transcription reaction, and has been carefully optimized so that 3'-end poly(A) modification and reverse transcription proceed efficiently at the same time. For downstream qPCR, only a specific forward primer needs to be designed and combined with the universal reverse primer supplied in the kit to detect miRNAs in the sample. Universal U6 reference forward and reverse primers for human, rat, and mouse are also included and deliver well-behaved standard curves over a broad range. For optimal results, the kit is recommended for use with Alta DiagnoTech's miRNA Universal qPCR SYBR Master Mix. |
| Intended Use | First-strand cDNA synthesis from miRNA using the poly(A) tailing method, for downstream qPCR-based miRNA detection and quantification. |
| Principle / Technology | Poly(A) tailing method: a poly(A) tail is added to the 3' end of miRNA while reverse transcription proceeds simultaneously in the same optimized buffer system. Downstream qPCR uses a miRNA-specific forward primer together with the kit's universal reverse primer; U6 serves as an internal reference. |
| Detection Method | Reverse transcription followed by SYBR Green qPCR using a specific forward primer and the universal reverse primer. |
| Sample Type | Total RNA or miRNA samples; U6 reference primers are universal for human, rat, and mouse samples. |
| Performance Range / Specifications | Reverse transcription: 37°C for 50 min; inactivation: 85°C for 5 sec. Sensitivity of 10 pg with total RNA template and no fewer than 60 copies with miRNA template. Reagent stable at 37°C for 7 days and through 50 freeze-thaw cycles; prepared reaction mix retains performance after 48 h at 25°C. |
| Sensitivity / LOD | Sensitivity of 10 pg when total RNA is used as template; no fewer than 60 copies when miRNA is used as template. |
| Specificity | High specificity; distinguishes single-base differences among members of the same miRNA family (validated on hsa-let-7 family subtypes hsa-miR-let-7a to let-7i and hsa-miR-98). |
| Reaction Conditions / Protocol | Reverse transcription at 37°C for 50 min; enzyme inactivation at 85°C for 5 sec. |
| Components / Formulation | miRNA RT Enzyme Mix: 17.5 μL (10 T) / 87.5 μL (50 T) 2x miRNA RT Buffer: 50 μL (10 T) / 250 μL (50 T) RNase-free H2O: 2 mL (10 T) / 10 mL (50 T) Universal Reverse Primer (10 μM): 800 μL (10 T) / 4 mL (50 T) U6 Forward Primer (10 μM): 100 μL (10 T) / 500 μL (50 T) U6 Reverse Primer (10 μM): 100 μL (10 T) / 500 μL (50 T) |
| Storage Conditions | Store at -20°C. |
| Shelf Life | 1 year when stored at -20°C. |
| Package Specifications | 10 T, 50 T |
| Product Form | Kit |
| Quality Control | Reagent stable at 37°C for 7 days and through 50 freeze-thaw cycles; the prepared reaction system retains performance after 48 h at 25°C. |
| Concentration | Universal Reverse Primer: 10 μM; U6 Forward Primer: 10 μM; U6 Reverse Primer: 10 μM |
| Shipping Conditions | Shipped with ice packs. |
| Expiration Date / Stability | Valid for 1 year at -20°C; reagent stable at 37°C for 7 days and through 50 freeze-thaw cycles; prepared reaction mix stable for 48 h at 25°C. |
| Compatibility | Recommended for use with Alta DiagnoTech's miRNA Universal qPCR SYBR Master Mix for optimal results. Common general-purpose qPCR reagents typically only amplify 80-300 bp fragments and cannot amplify the short miRNA products; a dedicated miRNA qPCR reagent ensures amplification of these short fragments. |
| Application Notes / Precautions | The sequence of the universal downstream reverse primer is proprietary: poly(A) tailing reagents on the market add different A-tailed sequences designed around their own tailing enzymes, and the sequence is protected. The U6 primers target the miRNA internal reference U6 for downstream quantitative detection. With the poly(A) tailing design, a single reverse transcription product can be used to detect multiple miRNAs: only a specific forward primer needs to be designed for each target and paired with the universal reverse primer. Use a dedicated miRNA qPCR reagent for downstream quantification, as ordinary qPCR reagents generally cannot amplify the short cDNA fragments generated from miRNA. |
For research use only, not for clinical use.
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