cDNA Synthesis Kit, Oligo(dT) and Random Hexamer Primers, 5x All-In-One Master Mix, RNase H+

Name: cDNA Synthesis Kit, Oligo(dT) and Random Hexamer Primers, 5x All-In-One Master Mix, RNase H+
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Product Name	cDNA Synthesis Kit, Oligo(dT) and Random Hexamer Primers, 5x All-In-One Master Mix, RNase H+
Catalog No.	NATR-HMM-0155
Description	All-in-one 5x concentrated cDNA synthesis master mix combining M-MLV reverse transcriptase (RNase H+), optimized buffer, dNTPs, oligo(dT)18 and random hexamer primers, and RNase inhibitor in a single tube for streamlined first-strand cDNA synthesis. Designed for fast and convenient conversion of total RNA or poly(A)+ mRNA into cDNA for downstream qPCR and conventional PCR applications. The unique 5x all-in-one RT master mix format reduces pipetting steps, minimizes contamination risk, and eliminates the need for separate primer selection. The mix combines oligo(dT) primers (which prime from the poly(A) tail of mRNA, favoring the 3' end) with random hexamers (which prime throughout the transcript, providing better coverage of 5' regions and long transcripts, and also prime non-polyadenylated RNA including some lncRNA and viral RNA). The M-MLV RT (RNase H+) retains its intrinsic RNase H activity, which degrades the RNA strand in RNA-DNA hybrids after first-strand synthesis — this is beneficial for qPCR as it reduces RNA interference with subsequent PCR amplification but may slightly reduce full-length cDNA yield for very long transcripts (>5 kb).
Intended Use	First-strand cDNA synthesis from total RNA or mRNA for: two-step RT-qPCR gene expression analysis; conventional RT-PCR; cDNA library construction; rapid screening by PCR; and standard molecular biology applications. The all-in-one format is ideal for high-throughput applications where simplicity, speed, and reproducibility are priorities.
Principle / Technology	M-MLV reverse transcriptase catalyzes RNA-dependent DNA synthesis using the combined oligo(dT)18 and random hexamer primers. Oligo(dT)18 primes from the poly(A) tail at the 3' end of mRNA. Random hexamers prime at multiple sites throughout the RNA, producing shorter cDNA fragments that cover the entire transcript length including 5' ends. The 5x master mix contains all components at optimized concentrations: RT enzyme, dNTPs (0.5 mM each in 1x), primers (optimized blend), RNase inhibitor, MgCl2, and buffer. The user only adds RNA template and nuclease-free water. The RNase H activity of the RT enzyme degrades the template RNA after cDNA synthesis, reducing its potential to interfere with qPCR.
Detection Method	1) Thaw 5x Master Mix on ice, mix gently; 2) Prepare reaction: 4 uL 5x Master Mix, 1 pg-2 ug total RNA (or 1 pg-500 ng mRNA), nuclease-free water to 20 uL; 3) Incubate at 25 C for 10 min (random hexamer annealing and extension), then 42 C for 30-60 min (cDNA synthesis), then 85 C for 5 min (enzyme inactivation); 4) Dilute cDNA 1:5 to 1:20 in nuclease-free water and use 1-2 uL per qPCR reaction.
Sample Type	Total RNA (1 pg-2 ug per 20 uL reaction), poly(A)+ mRNA (1 pg-500 ng), in vitro transcribed RNA, viral RNA. RNA quality: A260/A280 1.8-2.1, RIN >=7 for best results. RNA must be dissolved in nuclease-free water or TE; avoid DEPC-treated water (residual DEPC may inhibit RT).
Performance Range / Specifications	RNA input range: 1 pg-2 ug total RNA (7 orders of magnitude linear range); cDNA synthesis efficiency: >80% RNA-to-cDNA conversion (based on qPCR Ct comparison); reaction time: 40-70 min total (includes 10 min hexamer annealing, 30-60 min RT, 5 min inactivation); cDNA length: effective synthesis up to 8 kb (reduced yield for >5 kb due to RNase H activity).
Sensitivity / LOD	cDNA from as little as 1 pg total RNA (approximately 0.1 cell equivalents) amplifiable by qPCR; linear Ct vs log(input RNA) over 6-7 orders of magnitude.
Specificity	Combined oligo(dT) and random hexamer primers capture all polyadenylated mRNA (via dT priming) and also transcribe non-polyadenylated RNA and internal regions of long transcripts (via random hexamers). No significant genomic DNA reverse transcription (M-MLV RT is RNA-dependent DNA polymerase; DNA-dependent activity is ~10x lower at standard RT conditions). However, genomic DNA contamination in RNA will be carried over to downstream PCR — include a no-RT control for each RNA sample.
Reaction Conditions / Protocol	Step 1: 25 C, 10 min (random hexamer annealing/extension); Step 2: 42 C, 30-60 min (cDNA synthesis; 30 min sufficient for most applications; 60 min for low-abundance transcripts or GC-rich RNA); Step 3: 85 C, 5 min (RT inactivation).
Components / Formulation	5x All-In-One cDNA Synthesis Master Mix (400 uL total, 2 x 200 uL vials): M-MLV Reverse Transcriptase (RNase H+), optimized RT buffer, dNTPs (0.5 mM each in 1x), oligo(dT)18 primers, random hexamer primers, recombinant RNase inhibitor, MgCl2, stabilizers; Nuclease-Free Water (1.5 mL); Protocol.
Storage Conditions	5x Master Mix at -20 C; stable at 2-8 C for up to 1 month; avoid repeated freeze-thaw cycles (>10 tolerated); protect from light.
Shelf Life	24 months at -20 C.
Package Specifications	400 uL 5x Master Mix (sufficient for 100 reactions at 20 uL); 200 reactions at 10 uL half-reaction scale.
Product Form	Frozen liquid; thawed: clear, colorless to pale yellow viscous liquid.
Quality Control	Each lot: RT efficiency: >80% RNA-to-cDNA conversion (qPCR Cq shift <1 vs ideal 100% conversion); linear dynamic range: R2 >0.99 over 1 pg-2 ug total RNA input (GAPDH qPCR); no-RT control: no amplification (Ct >35 or undetermined); DNase, RNase: negative; functional test: cDNA from 1 ug HeLa total RNA amplifies GAPDH, ACTB, and 18S rRNA in qPCR (Ct <=20); lot-to-lot CV of cDNA yield <10%.
Key Features	5x all-in-one master mix format; single-tube setup (just add RNA + water); combined oligo(dT) and random hexamer primers; RNase inhibitor included; 7-log linear dynamic range; 40-70 min protocol; suitable for 1 pg-2 ug RNA; 100-reaction kit.
Purity	M-MLV RT: >95% by SDS-PAGE; DNase, RNase negative; RNase inhibitor: recombinant (E. coli-expressed, animal-free); dNTPs: >99% HPLC; no host DNA or RNA detected.
Concentration	5x Master Mix; 1x working concentration in 20 uL reaction.
Activity / Unit Definition	M-MLV RT: approximately 200 U per 20 uL reaction; 1 U = amount incorporating 1 nmol dTMP in 10 min at 37 C using poly(A)-oligo(dT) template.
Molecular Weight	M-MLV RT: ~75 kDa (monomer); RNase inhibitor: ~50 kDa.
Source / Origin	M-MLV RT: recombinant, E. coli-expressed; RNase inhibitor: recombinant (E. coli-expressed); dNTPs: synthetic; primers: synthetic oligonucleotides; animal-free production.
pH Range / Optimal pH	Master mix pH 8.3 (at 25 C); optimal for M-MLV RT activity.
Shipping Conditions	Dry ice (-20 C); stable at ambient for up to 3 days transit.
Expiration Date / Stability	24 months at -20 C; 1 month at 2-8 C; 10 freeze-thaw cycles tolerated.
Regulatory / Compliance	For research use only; not for diagnostic or therapeutic use. ISO 9001 certified; RNase-free certified.
Compatibility	cDNA product compatible with all standard Taq and high-fidelity DNA polymerases. Compatible with SYBR Green and probe-based (TaqMan) qPCR master mixes. cDNA can be used directly in PCR without purification — typical input: 1-2 uL cDNA per 20-25 uL qPCR. For low-abundance transcripts, use up to 4 uL cDNA (10-20% of qPCR reaction). Compatible with standard and fast qPCR cycling protocols. RNA template must be free of: phenol (inhibits RT), guanidine (carryover from TRIzol extraction), EDTA at >1 mM (chelates Mg2+), and SDS at >0.01%. Ethanol carryover from RNA precipitation should be removed by air-drying or speed-vac before dissolving RNA.
Recommended Buffer System	5x Master Mix: Tris-HCl pH 8.3, KCl, MgCl2 (optimized), dNTPs, oligo(dT)18, random hexamers, M-MLV RT, RNase inhibitor, stabilizers (glycerol, BSA, detergent).
Application Notes / Precautions	Thaw the 5x Master Mix on ice and mix gently by pipetting or brief vortex before use (viscous solution). Prepare the RT reaction on ice. Include a no-RT control (replace 5x Master Mix with nuclease-free water in a parallel reaction containing the same RNA; this control reveals genomic DNA contamination in downstream PCR). For RNA with high secondary structure (GC-rich, structured UTRs), preheat RNA at 65 C for 5 min and chill on ice immediately before adding to reaction. For very long transcripts (>5 kb), use 60 min RT incubation or consider an RNase H-minus (point mutant) RT for better full-length synthesis. After cDNA synthesis, dilute 1:5 to 1:20 before qPCR. Standard dilution 1:10 for most applications (add 180 uL water after 20 uL RT reaction). Store cDNA at -20 C (short term, <1 month) or -80 C (long term).
Batch-to-Batch Consistency	RT efficiency >80% for every lot; linear dynamic range R2 >0.99; no-RT control negative; DNase/RNase negative.

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For research use only, not for clinical use.