Performance of RTPCR kits for reproducible production of cDNA libraries

Introduction: To quantify RNA, the RNA sample must first be converted into cDNA, a form less susceptible to degradation than RNA, through the process of reverse transcription (RT). The enzyme that converts RNA to cDNA, reverse transcriptase, has many modified forms. If the RT process does not result in reproducible cDNA libraries that represent all message species in the RNA extract, trail conclusions may contain errors. Reproducible data is mandatory for accuracy in experiments relying on RNA quantification.

Research Question: Which RTPCR kits produce the most cDNA?

Methods: An extraction of RNA from dried blood samples was used as a template for testing the effectiveness of different RTPCR kits (Qiagen QuantiTech (Q), BioRad iScript (I), Thermo Fischer SuperScript IV (SSIV or S), and New England LunaScript (N)). Once the kits were utilized on equal amounts of RNA, the cDNA samples were put into the PCR to determine the quantity of cDNA present.

Results: All kits showed high reproducibility among the triplicates. Overall, the SSIV kit gives the highest yield of cDNA, indicated by lower Ct values. The New England LunaScript kit gives the lowest yield of cDNA, indicated by higher Ct values.

Conclusion: The results suggest that the SSIV RT enzyme produces the most cDNA and also occurs in a short reaction time; a fast enzyme reaction suggests high efficiency in the SSIV modification of RT. On the other hand, this experiment indicates that the least efficient RT modification is the N kit due to its low yield of cDNA.