qPCRBIO cDNA Synthesis Kit is an easy-to-use 2 tube system specifically developed to generate cDNA for use in real-time PCR.
High quality cDNA synthesis is essential for downstream real-time PCR analysis and successful expression studies.
The reverse transcriptase, buffer system and optimised blend of random hexamers with anchored oligo(dT) primers provide unbiased, efficient and sensitive cDNA synthesis over a broad range of RNA template concentrations.
- Unbiased representation of 5’ and 3’ mRNA transcript ends
- Sensitive detection of low copy number transcripts
- High cDNA yields from as little as 4pg total RNA
- Reduced RNase H activity
- Simple 2 tube system
- 5x buffer contains anchored oligo(dT), random hexamers, enhancers, dNTPs and MgCl2
- 20x thermostable reverse transcriptase blended with RNase inhibitor
qPCRBIO cDNA Synthesis Kit uses the latest developments in reverse transcriptase technology and buffer chemistry to enhance cDNA synthesis speed, yield and representation. There are many important considerations when performing cDNA synthesis such as the priming strategy, reverse transcriptase, buffer and enhancers used. qPCRBIO cDNA Synthesis Kit removes the need for user optimisation of these critical factors to give unbiased, efficient and sensitive cDNA synthesis.
The modified MMLV reverse transcriptase (RTase) is both thermostable and extremely active. The enzyme is blended with RNase inhibitor preventing degradation of RNA by contaminating RNase. The RTase is not inhibited by ribosomal and transfer RNAs, making total RNA an ideal substrate. The 5x cDNA synthesis mix can be used with up to 0.4μg total RNA.
The relative concentrations of random hexamers and anchored oligo(dT) have been optimised for the generation of cDNA for use in real-time PCR experiments.
- cDNA synthesis for real-time PCR analysis
- Low copy number transcripts
- Viral RNA targets
- Efficient synthesis from total RNA or poly(A)+ RNA
The kit should be stored between -30°C and -15°C. Avoid prolonged exposure to light. If stored correctly the kit will retain full activity for 12 months. The kit can be stored at 4°C for 1 month. The kit can go through 30 freeze/thaw cycles with no loss of activity.
Figure 1. Broad reverse transcription dynamic range
qPCRBIO cDNA Synthesis Kit was used for cDNA synthesis using a 10 fold serial dilution of mouse total RNA from 40pg to 400ng. qPCR was performed using qPCRBIO SyGreen Mix amplifying a 122bp fragment of the mouse ACTG gene. Efficiency was measured at 96% across the range tested.
The results demonstrate that qPCRBIO cDNA Synthesis Kit efficiently reverse transcribes RNA across a broad dynamic range of substrate.
Figure 2. Unbiased representation of mRNA ends
a) qPCRBIO cDNA Synthesis Kit was used to synthesise cDNA from mouse liver total RNA. 2 primer pairs were designed against the 5’ (red traces) and the 3’ (black traces) ends of the 4.2kb mouse CANX transcript. qPCRBIO SyGreen Mix was used for analysis. The primer pairs were 4kb apart and did not show any reverse transcription bias, hence the amplification traces overlap.
b) 2 primer pairs against the 5’ (red) and 3’ (black) traces of RNS18 gene (1.8kb). Again, no reverse transcription bias was evident.
Figure 3. Thermostable enzyme for high GC%
a) qPCRBIO cDNA synthesis kit was used to synthesise cDNA from mouse liver total RNA at 42˚C (red) and also at 55˚C (black). A primer pair was designed against GJB2, generating an 84% GC amplicon. qPCRBIO SyGreen Mix was used for analysis. The higher temperature incubation generated more GC rich cDNA than the low temperature incubation.
b) A control amplicon of 55% GC from GAPDH was amplified from the 2 cDNAs described above. For this GC% no advantage of higher temperature incubation was achieved. The yield was slightly lower with the higher temperature.
|PB30.11-021||25 x 20μL Reactions|
|PB30.11-10||100 x 20μL Reactions|
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|20x RTase with RNase Inhibitor||
25 Reactions: 1 x 25μL
100 Reactions: 1 x 100μL
|5x cDNA Synthesis Mix||
25 Reactions: 1 x 100μL
100 Reactions: 1 x 400μL