The TRANSCRIPTME RNA Kit is a system which includes all the necessary components for synthesizing first-strand cDNA, apart from the template total RNA or mRNA. The synthesized single-stranded cDNA is suitable for real-time quantitative RT-PCR applications. The TRANSCRIPTME RNA Kit has been formulated to provide high yields of full-length cDNA product and to increase sensitivity in RT-qPCR. Starting material can range from 10 pg to 2.5 μg of total RNA.
The kit includes both the TRANSCRIPTME Enzyme Mix, with a recombinant thermostable reverse transcriptase M-MuLV without RNase H activity and the RIBOPROTECT RNase Inhibitor. The optimal reaction buffer with a combination of random hexamers and oligo(dT)18 primers, MgCl2 and dNTPs mix is also provided for increased sensitivity.
TRANSCRIPTME Reverse Transcriptase 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 which are rich in GC pairs and/or contain secondary structures. The enzyme has no 3’–5’ exonuclease or RNase H activity, which improves 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.
Use of the recombinant RNase inhibitor RIBOPROTECT protects the RNA template from degradation. The enzyme selectively hydrolyses the phosphodiester bonds of RNA only when it is hybridized to 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 reaction since the PCR primers will bind more easily to the cDNA. Using of the RNase H is recommended only when it contributes to a full-length cDNA synthesis and increased yields of first -strand cDNA.
Features and advantages
- High yields of full-length cDNA products (up to 10 kb)
- Formulated to increase sensitivity in RT-qPCR and RT-PCR assays
- Fewer pipetting steps – minimized contamination risk
- Simultaneous cDNA synthesis with mRNA and rRNA templates
- Starting material: 10 pg – 5 μg of total RNA or 10 pg – 500 ng of mRNA
- Optimal reaction temperature: 50°C
- The reverse transcriptase shows no RNase H or 3’-5’ exonuclease activity, has supreme thermostability and is suitable for difficult RNA templates
- RNase inhibitor protects the RNA template from degradation
- Additional RNse H treatment may increase the sensitivity of the RT-qPCR reaction
- Full-length cDNA template synthesis for RT-qPCR and two-step RT-PCR assays
- cDNA synthesis for molecular cloning
- cDNA library construction
- RNA analysis
We recommend the EXTRACTME TOTAL RNA KIT (EM09) and EXTRACTME TOTAL RNA PLUS KIT (EM11) for total RNA isolation from tissues and cell cultures
During RT-PCR reaction mixture preparation, keep the TRANSCRIPTME Reverse Transcriptase and 10x RT Reaction Buffer on ice or in a freezing rack
Use an RNase H treatment for reactions sensitive to residue RNA traces in order to increase the sensitivity of the RT-qPCR
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 TRANSCRIPTME Reverse Transcriptase is inhibited by metal ion chelating agents (e.g. EDTA), inorganic phosphor, pyrophosphate and polyamines
Working with RNA
Acquisition of high quality, intact RNA, free of genomic DNA and RNase traces, is vital for the synthesis of a full-length cDNA followed by an accurate quantitative analysis (qPCR). The following recommendations for working with RNA should therefore be followed:
Maintain aseptic working conditions: use disposable gloves, changing them as frequently, as required; use RNase-free consumables; only work in an area assigned for working with RNA and with equipment designated for that purpose.
DNase I enzyme (not included) may be used if obtaining a DNA-free RNA sample is required.
RNA samples should be stored aliquoted at -70°C. Avoid subjecting the samples to repeated freezing and thawing cycles.
Store all components at -20°C. The stability of the reagents can be extended if stored at -70°C. The product is stable for 12 months providing it is stored and used correctly.
Shipping on dry ice
Manufacturer: BLIRT S.A.