When synthesizing a protein, DNA is transcribed into mRNA which is then translated into a protein. One difference between eukaryotic and prokaryotic genes is that eukaryotic genes often contain introns which are not coding sequences, in contrast with exons, which are DNA coding sequences. During transcription, intronic RNA is excised from the RNA primary transcript and the remaining pieces of the RNA primary transcript are spliced back together resulting in processed mRNA. The mRNA code is then translated into an amino acid chain that comprises the newly made protein.
Using reverse transcriptase polymerases, DNA can be synthesised from mRNA and total RNA. Thus it is a 'complementary' copy of the mRNA, and is thus called complementary DNA (cDNA). cDNA forms the substrate for the majority of qPCR gene expression experiments.
qPCRBIO cDNA Synthesis Kit
- Unbiased representation of 5' and 3' mRNA transcript ends
- Sensitive detection of low copy number transcripts
- High cDNA yields from as little as 1pg total RNA
- Simple 2 tube system
The qPCRBIO cDNA Synthesis Kit uses the latest developments in reverse transcriptase technology and buffer chemistry to enhance cDNA synthesis speed and yield with accurate transcript representation. The reverse transcriptase, buffer system and combination of random hexamers with anchored oligo(dT) allow for unbiased, efficient, sensitive cDNA synthesis.
High quality cDNA synthesis for downstream qPCR analysis is essential for successful expression studies. Many factors affect cDNA synthesis including the reverse transcripase, buffer systems, enhancers and priming strategy. The PCRBIO cDNA synthesis mix removes the need for user optimisation of these critical factors.
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, total RNA is an ideal substrate. The 5x cDNA synthesis mix can be used with up to 4.0μg 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
- miRNA targets
- Efficient synthesis from total RNA or poly(A)+ RNA