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| Product Name | Random Hexamer Primers (50 μM) |
| Catalog No. | NATR-HMM-0136 |
| Description | A ready-to-use solution of random hexanucleotide primers for non-specific priming of reverse transcription reactions and DNA labeling procedures. The random sequence composition ensures unbiased priming across all RNA sequences, enabling comprehensive cDNA synthesis from the entire transcriptome. |
| Intended Use | Initiation of reverse transcription for first-strand cDNA synthesis from total RNA or mRNA; random priming for radiolabeled and fluorescent DNA probe synthesis by Klenow fragment; priming for second-strand cDNA synthesis; and random amplification of DNA for whole-genome amplification protocols. |
| Principle / Technology | A mixture of all possible hexanucleotide sequences (4^6 = 4,096 combinations) anneals to complementary sequences across the entire RNA template. Reverse transcriptase extends from these multiple priming sites, generating cDNA fragments that collectively represent the full transcript length distribution with improved 5ʹ-end coverage compared to oligo-dT-only priming. |
| Detection Method | cDNA yield quantitation by fluorometry or UV spectrophotometry; cDNA size distribution by Bioanalyzer or agarose gel electrophoresis; coverage uniformity assessed by RT-qPCR with 5ʹ and 3ʹ primer sets targeting different regions of reference transcripts |
| Sample Type | Total RNA from all sources (mammalian, plant, bacterial, yeast, viral); purified mRNA; in vitro transcribed RNA; degraded or partially fragmented RNA (random priming essential for fragmented samples); small RNA preparations |
| Performance Range / Specifications | Primer concentration: 50 μM in nuclease-free water or TE buffer; typical usage: 0.5–5 μM final concentration (50–250 ng per 20 μL cDNA synthesis reaction); random sequence composition verified by mass spectrometry analysis of nucleotide incorporation frequencies; DNase/RNase-free; HPLC-purified oligonucleotide synthesis quality |
| Sensitivity / LOD | 5ʹ-end coverage: detects transcript 5ʹ ends within 100 nucleotides for >90% of transcripts when used at 5 μM final concentration; cDNA yield: 2–5× higher than oligo-dT priming alone with total RNA input |
| Specificity | Sequence-randomness verified by next-generation sequencing of primed cDNA libraries; all 4,096 hexamer sequences represented within 10-fold of mean frequency; unbiased against GC-rich or AT-rich sequences |
| Reaction Conditions / Protocol | Combine RNA template with random hexamers (add 1–2 μL of 50 μM stock per 20 μL reaction), dNTPs, and nuclease-free water; denature at 65°C for 5 minutes, chill rapidly on ice; add RT buffer, RNase inhibitor, and reverse transcriptase; incubate at 25°C for 5–10 minutes (primer annealing), followed by 42–50°C for 30–60 minutes (cDNA synthesis); heat inactivate at 70°C for 10–15 minutes |
| Components / Formulation | Random hexanucleotide mixture (all 4,096 possible hexamer sequences, chemically synthesized and HPLC-purified), supplied at 50 μM in nuclease-free water or TE buffer (10 mM Tris-HCl pH 8.0, 1 mM EDTA); individual vials or bulk aliquots available |
| Storage Conditions | –20°C for long-term storage; stable at 2–8°C for up to 1 month; avoid repeated freeze-thaw (>20 cycles); aliquot into single-use volumes to prevent contamination and degradation |
| Shelf Life | 24 months at –20°C from date of manufacture |
| Package Specifications | 5 μg (1 vial at 50 μM), 25 μg, and 100 μg packaging |
| Product Form | Ready-to-use liquid solution; no reconstitution required |
| Quality Control | Each lot tested: RT-qPCR cDNA yield from 1 μg HeLa total RNA (GAPDH Ct ≤20); 5ʹ/3ʹ ratio ≤3 for GAPDH and ACTB transcripts by qPCR; random-sequence composition verified by mass spectrometry; DNase/RNase-free validation; no PCR inhibition in downstream qPCR with 10% cDNA input |
| Key Features | Ready-to-use 50 μM concentration eliminates dilution calculations; HPLC-purified for maximum cDNA synthesis efficiency; superior 5ʹ-end gene coverage compared to oligo-dT alone; essential for degraded RNA samples where poly(A) tails are fragmented; compatible with all common reverse transcriptases |
For research use only, not for clinical use.
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