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| Product Name | Random Hexamer Primer (dN6) for cDNA Synthesis |
| Catalog No. | PRIPRO-0019 |
| Description | A pre-synthesized random hexamer primer composed of all possible six-nucleotide sequence combinations (4⁶ = 4096 unique sequences), supplied as an equimolar mixture. Random hexamers are the most versatile primers for first-strand cDNA synthesis, capable of priming reverse transcription from any RNA template regardless of sequence, secondary structure, or polyadenylation status. This universal priming approach is essential for preparing cDNA libraries for qPCR gene expression profiling, RNA-seq library construction, and whole-transcriptome amplification. The oligo mixture is synthesized using a randomized phosphoramidite coupling strategy and purified by desalting or HPLC. Supplied as a lyophilized or pre-dissolved solution at a convenient concentration for immediate use in reverse transcription reactions. |
| Intended Use | Designed for first-strand cDNA synthesis from total RNA or mRNA in reverse transcription reactions preceding qPCR, conventional PCR, RNA-seq library construction, and whole-transcriptome amplification workflows. |
| Principle / Technology | Equimolar mixture of all 4096 hexamer sequences; each hexamer can prime reverse transcriptase at multiple sites along RNA templates, generating overlapping cDNA fragments for uniform transcript coverage |
| Detection Method | cDNA synthesized using MMLV or AMV reverse transcriptase; random priming produces cDNA fragments spanning the entire transcript; uniformity assessed by 5′/3′ ratio in qPCR |
| Sample Type | Total RNA extracted from cells, tissues, blood, or any biological source; applicable to eukaryotic, prokaryotic, and viral RNA |
| Performance Range / Specifications | Hexamer mixture (N₆) covering all 4096 sequences; typical use 50–250 ng per 20 µL reverse transcription reaction; primes mRNA, rRNA, tRNA, lncRNA, and viral RNA |
| Sensitivity / LOD | Enables reverse transcription from as little as 1 pg total RNA when paired with sensitive reverse transcriptase; successful cDNA synthesis from single-cell RNA quantities reported |
| Specificity | Non-specific priming by design; all RNA species are reverse transcribed; ideal for unbiased transcriptome coverage; may generate ribosomal RNA-derived cDNA, which should be accounted for in downstream analysis |
| Reaction Conditions / Protocol | Use 50–250 ng random hexamers per 20 µL RT reaction. Incubate with RNA at 65 °C for 5 min, chill on ice, add RT master mix, incubate at 25 °C for 10 min (annealing), then 42–50 °C for 30–60 min (extension), followed by 70 °C for 15 min (inactivation). |
| Components / Formulation | Lyophilized random hexamer mixture or pre-dissolved in nuclease-free water at 50–100 ng/µL (approximately 25–50 µM) |
| Storage Conditions | –20 °C; stable at –20 °C in solution; minimize freeze-thaw cycles |
| Shelf Life | 24 months at –20 °C; 12 months at 2–8 °C |
| Package Specifications | Tube containing 5–50 µg total hexamer; supplied lyophilized or in solution at 50–100 ng/µL |
| Product Form | Lyophilized white powder or colorless solution when dissolved |
| Quality Control | HPLC or PAGE profile showing uniform length distribution centered at 6 nt; functional testing: confirmed cDNA synthesis from 1 µg HeLa total RNA with >50% 5′/3′ coverage ratio for GAPDH; absence of detectable DNase and RNase activity. |
| Key Features | Universal RNA priming regardless of sequence; equimolar hexamer mixture for unbiased coverage; compatible with all commercial reverse transcriptases; suitable for single-cell and low-input RNA applications; functional QC validated by cDNA synthesis test. |
| Purity | ≥90% hexamer species by PAGE; uniform length distribution; free of truncated (<5 nt) and longer (>8 nt) contaminants |
| Concentration | 50 ng/µL = ~25 µM (typical); or 100 ng/µL = ~50 µM; verified by A260 measurement |
| Activity / Unit Definition | Efficiently primes cDNA synthesis by MMLV, AMV, and SuperScript reverse transcriptases; primers bound per kilobase of RNA: approximately 1 hexamer per 4 kb at recommended concentration |
| Molecular Weight | ~1,800–1,900 Da average (mixture of 4096 hexamers; each ~1,800 Da) |
| Source / Origin | Synthetic; randomized phosphoramidite coupling at each position; no biological raw materials |
| pH Range / Optimal pH | Stable and functional in reverse transcription buffer pH 7.5–8.5 |
| Shipping Conditions | Ambient temperature; lyophilized or frozen solution on dry ice |
| Expiration Date / Stability | 24 months at –20 °C; 12 months at 2–8 °C; avoid repeated freeze-thaw cycles of aliquots |
| Regulatory / Compliance | For research use only; manufactured under ISO 9001 quality system; not for diagnostic or therapeutic use |
| Compatibility | Compatible with all major reverse transcriptases: MMLV RT, AMV RT, SuperScript II/III/IV, Maxima RT, PrimeScript RT, and commercial cDNA synthesis kits |
| Recommended Buffer System | Nuclease-free water; compatible with all commercial RT buffer systems (Tris-HCl pH 8.3, KCl, MgCl₂, DTT) |
| Application Notes / Precautions | For RNA with strong secondary structure, increase hexamer concentration to 250 ng per reaction. For single-cell RNA-seq, use 5–50 ng per reaction. Hexamer priming generates shorter cDNA fragments than oligo-dT priming; ideal for fragmented or partially degraded RNA (e.g., FFPE samples). |
| Batch-to-Batch Consistency | Length distribution profile consistent across batches; cDNA synthesis efficiency within ±15% by qPCR GAPDH Ct; hexamer concentration within ±10% |
For research use only, not for clinical use.
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