In RNA-based research applications, reverse transcription (RT) is a very critical and crucial step. By utilizing proper RT reagents, RNA can be transcribed into single-stranded cDNA, making PCR and other DNA-based analyses possible. Such analyses are important in helping researchers more precisely assess how changes in the amount of RNA contribute to the development of disease or how they related to biological processes in cells. The solution from Applied Biosystems (High Capacity cDNA Reverse Transcription Kit) is a remedy for all such applications. These reagents are specifically validated and optimized to archive cDNA for gene quantification and expression studies.
Applied Biosystems High Capacity cDNA Reverse Transcription Kit uses random primers for reverse transcription of RNA into cDNA. The generated cDNA does not require any further clean up step because an incubation step at 85°C for 5 seconds inactivates all components of RT step (primers and enzyme), hence the reaction doesn’t require any clean up process and can be directly used for downstream applications like PCR, qPCR, Northern blotting, and microarray. The reason this kit is called “high capacity” is because 2 µg of total RNA can be converted in a single 20 µl reaction volume. The reaction can be scaled up to 100 µl to generate 10 µg of cDNA.
Each kit contains following components; 10X RT buffer (1.0 ml); 10X RT random primers (1.0 ml); 25X dNTP mix (100 mM) (0.2 ml); MultiScribe Reverse Transcriptase 50 U/ul (0.1 X 2 ml) and RNase inhibitor (100 ul). The reagents supplied are sufficient for 200 reactions (20 ul/reaction). Preparing a master mix requires combining 2.0 µl 10X RT buffer, 0.8 µl 25X dNTP mix, 2.0 µl 10X RT random primers, 1.0 µl Reverse Transcriptase, 1.0 µl RNase inhibitor, 3.2 µl nuclease-free water and 10.0 µl RNA. Total final volume is 20 µl.
Thermal profiling:
Step 1 - 25 °C for 10 minutes
Step 2 - 37 °C for 120 minutes
Step 3 - 85 °C for 5 seconds
Step 4 - 04 °C for ∞
We use this kit routinely for the gene expression studies where we convert total RNA into cDNA in association with real-time PCR as a downstream application. We use 1 µg of total RNA (using QIAamp RNA Blood Mini Kit from Qiagen) isolated from leukocytes (source: whole blood) and convert it into cDNA. This cDNA reverse transcription kit is an excellent tool for any kind of RT application. The only thing lacking is an oligo d(T) primer which would allow the end user can’t greater flexibility.