Cat. No. / ID: RT31-020
The Reverse Transcription Kit contains all the necessary components for synthesizing first-strand cDNA from the total RNA or mRNA templates. The synthesized single-stranded cDNA is suitable for real-time quantitative RT-PCR applications. The kit provides high yields of full-length cDNA products and increases RT-qPCR sensitivity. Starting material can range from 10 pg to 5 μg of total RNA.
The kit includes a recombinant thermostable reverse transcriptase M-MuLV without RNase H activity and the RNase Inhibitor HU. The optimal reaction buffer with a combination of random hexamers, oligo (dT)18 primers, and MgCl2 and dNTPs mix increases sensitivity.
Reverse Transcription Kit has increased thermal stability, which allows the reaction to be carried out at a higher temperature (optimum activity at 50°C). It increases the efficiency and specificity of those transcribed RNA regions rich in GC pairs and containing secondary structures. The enzyme has no 3’→5’ exonuclease or RNase H activity, which improves the synthesis of a full-length cDNA, even from long mRNA templates, using random priming. The enzyme gives high yields of first-strand cDNA up to 10 kb long.
Using the recombinant RNase inhibitor HU protects the RNA template from degradation. RNase H selectively hydrolyzes the phosphodiester bonds of RNA only when hybridized into DNA. The RNase H does not degrade either single and double-stranded DNA or unhybridized RNA. This optional step can improve the sensitivity of subsequent RT-qPCR reactions since the PCR primers will bind more easily to the cDNA.
The RNase H is recommended only when it contributes to full-length cDNA synthesis and increased yields of first-strand cDNA.
Working with RNA
Acquiring high-quality, intact RNA, free of genomic DNA and RNase traces, is vital for synthesizing a full-length cDNA followed by an accurate quantitative analysis (qPCR).
The following recommendations should be followed:
During RT-PCR reaction mixture preparation, keep all kit reagents on ice or in a freezing rack.
Use an RNase H treatment for reactions sensitive to residue RNA traces to increase the RT-qPCR's sensitivity.
The quantity of cDNA used when preparing PCR or qPCR reactions should not exceed 1/10 of the final reaction volume; e.g., a maximum volume of 2.5 µL of cDNA should be used in a 25 µL reaction.
The activity of Reverse Transcriptase Kit is inhibited by metal ion chelating agents (e.g., EDTA), inorganic phosphor, pyrophosphate and polyamines.
Protein purity is determined using assay by SDS-PAGE electrophoresis resulting in >90% purity.
RNase and DNase contamination is evaluated by assessing RNase and DNase activity. In addition, functional quality is tested by RT-PCR experiment.
This is used for applications such as: