Technical Specification: Reverse Transcription Reagents¶

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Technical documentation published by Beijing Solarbio Science & Technology Co., Ltd. For product procurement and custom orders: solarbio.store | solarbio.store

1. Product Range¶

Product	SKU	Chemistry	Feature	Best For
First-Strand cDNA Synthesis Kit	PC1170	M-MLV RT (RNase H⁻)	Random hexamer + oligo-dT₁₈, 2-step RT	Gene expression, cloning, qPCR template
2×SYBR Green RT-qPCR Kit	SR1130	M-MLV RT + Hot-start Taq	One-step SYBR Green RT-qPCR	RNA quantification, fast workflow
2×TaqMan RT-qPCR Kit	SR1140	M-MLV RT + Hot-start Taq	One-step TaqMan RT-qPCR	RNA virus detection, multiplex

1.1 Key Advantages of Solarbio RT Reagents¶

Advantage	Details
RNase H⁻ mutant M-MLV	5× higher thermal stability than wild-type M-MLV; retains activity at 50°C for GC-rich RNA templates
Combined hexamer + oligo-dT priming	Uniform coverage across mRNA length; improved 5′ end representation vs. oligo-dT alone
RNase inhibitor included	Porcine RNase inhibitor (RNasin homolog) — protects RNA during reaction setup
One-step kits (SR1130/SR1140)	No separate cDNA step reduces hands-on time by 60 min; minimizes pipetting errors
Long cDNA synthesis	Full-length cDNA up to 7 kb (PC1170) with optimized extension time

2. Enzyme Specifications (PC1170)¶

2.1 M-MLV Reverse Transcriptase (RNase H⁻)¶

Parameter	Specification
Reverse transcriptase	M-MLV (RNase H⁻ mutant) — point mutation in RNase H domain
Molecular weight	76 kDa (monomer)
Optimal temperature	37–42°C standard; up to 50°C with GC-rich or structured RNA
Optimal pH	8.3 (Tris-HCl) at 37°C
DTT requirement	5 mM (included in 2× buffer)
Input RNA	10 pg – 5 μg total RNA; 1 pg – 500 ng poly(A)+ mRNA
Reaction time	30 min (standard), up to 60 min (GC-rich, long template)
Inactivation	70°C, 10 min (irreversible denaturation)
Primers supplied	Random hexamer (50 μM) + Oligo-dT₁₈ (50 μM) — 1:1 ratio in primer mix
Reaction volume	20 μL (standard), scalable to 100 μL
cDNA length	Up to 7 kb (with optimized 60 min extension)

2.2 The Reverse Transcription Reaction¶

The reverse transcription reaction catalyzed by M-MLV RT follows:

[ \text{RNA} + \text{Primer} \xrightarrow{\text{M-MLV RT, dNTPs, Mg}^{2+}} \text{RNA/DNA hybrid} + \text{PP}_i ]

The reaction is a three-step process:

Primer annealing (25°C, 10 min): Random hexamers and/or oligo-dT₁₈ hybridize to complementary sequences on the RNA template
Extension (42–50°C, 30–60 min): M-MLV RT extends the 3′-OH of the primer, incorporating dNTPs complementary to the RNA template. The RNase H⁻ mutation prevents degradation of the RNA template during first-strand synthesis, allowing full-length cDNA generation
Inactivation (70°C, 10 min): Heat denaturation inactivates the RT enzyme and dissociates the RNA/cDNA hybrid

2.3 Priming Strategy Comparison¶

Primer Type	Mechanism	Best For	5′/3′ Bias
Oligo-dT₁₈	Anneals to poly(A) tail of mRNA	Full-length mRNA, 3′ gene expression	3′ biased
Random hexamers (N₆)	Anneals to random complementary 6-mer sequences throughout all RNA species	Total RNA (including rRNA, viral RNA), 5′ coverage of long transcripts	Even coverage (5′-3′)
Gene-specific primers	Anneals to specific RNA sequence	Single-gene RT-qPCR, viral RNA detection	Target-specific
Combined (hexamer + oligo-dT)	Both mechanisms active simultaneously	Uniform 5′-3′ coverage, standard gene expression	Balanced (recommended default)

2.4 M-MLV (RNase H⁻) vs. Wild-Type M-MLV¶

Property	Wild-Type M-MLV	RNase H⁻ Mutant (PC1170)
RNase H activity	Present (degrades RNA in RNA/DNA hybrid)	<0.5% residual
Processivity	~150 nt	~350 nt
Optimal temperature	37°C	42°C (active to 50°C)
Half-life at 50°C	<5 min	~30 min
cDNA yield (from 5 kb template)	Reference (1×)	2–3× higher
Full-length cDNA (≥ 5 kb)	Variable	Consistent

3. Two-Step RT-qPCR Workflow¶

3.1 Recommended cDNA Synthesis Setup (PC1170)¶

Component	Volume (20 μL)	Final Concentration / Amount
Total RNA	Variable	10 pg – 5 μg (1 μg recommended for standard qPCR)
2× RT Buffer (includes dNTPs, DTT, Mg²⁺)	10 μL	1×
Primer Mix (random hexamer + oligo-dT₁₈)	1 μL	2.5 μM each
M-MLV RT (RNase H⁻)	1 μL	200 U
RNase inhibitor	0.5 μL	20 U
RNase-free water	To 20 μL	—

3.2 Thermal Protocol¶

Step	Temperature	Time	Purpose
Primer annealing	25°C	10 min	Allow random hexamers to anneal (not required for oligo-dT only)
Extension	42°C	30–60 min	First-strand cDNA synthesis
Inactivation	70°C	10 min	Heat-inactivate M-MLV RT and dissociate RNA/cDNA hybrid
Hold	4°C	∞

3.3 cDNA Dilution and Storage¶

Application	Recommended cDNA Dilution	Volume per qPCR (20 μL)
Standard gene expression (abundant targets)	1:10	2 μL
Low-expression targets	1:2–1:5	2–4 μL
Single-cell RT-qPCR	Undiluted	5 μL (max 10% of reaction volume)
Long-term storage	−20°C (≤ 6 months) or −80°C (≥ 2 years)	Aliquot to avoid freeze-thaw

3.4 No-RT Control¶

Always include a no-reverse-transcriptase (NRT) control for each RNA sample to assess genomic DNA contamination:

NRT Reaction	Expected Result	Interpretation
All components except M-MLV RT	Ct ≥ 35 or undetermined	No significant gDNA contamination
NRT Ct < 33	gDNA present	Treat RNA with DNase I; redesign primers to span introns

4. One-Step RT-qPCR Kits (SR1130/SR1140)¶

4.1 Reaction Setup¶

Component	Volume (20 μL)	Final
2× One-Step RT-qPCR Master Mix	10 μL	1×
Forward primer (10 μM)	0.4 μL	0.2 μM
Reverse primer (10 μM)	0.4 μL	0.2 μM
Probe (10 μM) — for TaqMan only	0.3 μL	0.15 μM
RNA template	1–5 μL	10 pg – 1 μg
RT enzyme mix	0.5 μL	—
ROX (if required)	0.4 μL	1×
RNase-free water	To 20 μL	—

4.2 One-Step Thermal Protocol¶

Step	Temperature	Time	Cycles
Reverse transcription	50°C	15 min	1
RT inactivation + Polymerase activation	95°C	2 min	1
Denaturation	95°C	10–15 s	40
Annealing/Extension (+ read)	60°C	30–45 s	40
Melt curve (SYBR only)	65–95°C, 5 s/step	—	1

4.3 One-Step vs. Two-Step RT-qPCR Comparison¶

Factor	One-Step (SR1130/SR1140)	Two-Step (PC1170 + SR1110)
Time to result	1.5 h	3–3.5 h
Hands-on time	10 min	30 min
Sensitivity	Equivalent	Equivalent
Multiplex targets	Up to 4 (TaqMan)	Up to 4 (TaqMan)
Replicate RNA samples in qPCR	No (single RNA per reaction)	Yes (aliquots of same cDNA)
Archival cDNA for future assays	No	Yes
Flexibility to test different genes later	Limited	High (cDNA bank)
RNA secondary structure handling	50°C RT, 15 min	42°C, 30–60 min (better for structured RNA)

5. Troubleshooting¶

Issue	Cause	Solution
No cDNA / high Ct in qPCR	RNA degraded	Check RNA integrity (gel/RIN); use fresh RNA
	RT enzyme inactive	Store at -20°C; avoid freeze-thaw; check expiry
	RNase contamination	Use fresh aliquots of RNase-free water; change gloves
	Inhibitor in RNA eluate	Reduce RNA input; dilute RNA 1:5; re-purify RNA
Low signal in downstream qPCR	RNA input too low	Increase RNA to 1–2 μg (PC1170); use 0.5 μg minimum
	Priming suboptimal	Use combined hexamer + oligo-dT; consider gene-specific primers
	GC-rich RNA fails to reverse transcribe	Increase RT temperature to 50°C; extend to 60 min
Late Ct in NRT control	Genomic DNA contamination	DNase treat RNA; use intron-spanning primers; include NRT control
Multiple melt peaks in SYBR RT-qPCR	Non-specific RT priming	Reduce primer concentration; increase RT temperature gradually
	gDNA amplification	Design primers spanning exon-exon junctions
	RNA degradation fragments	Check RNA quality; re-extract if necessary
cDNA sheared / < 1 kb	RNase contamination during setup	Use fresh gloves, sterile bench, DEPC-treated water
	Excessive heating at 70°C	Do not exceed 70°C for 10 min
	Prolonged storage at 4°C	Store cDNA at -20°C; avoid 4°C for > 24 h

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