Sepsis is one of the leading causes of patient mortality and is caused by an inflammatory response to infection. Identifying sepsis at an early stage can improve treatment methods and have a major positive impact on health conditions. The aim of this study was to compare two methods of total RNA extraction, manual vs robotic.
The comparison was done with hands-on time (HOT), turn-around time (TAT) and absolute quantification. The main focus was to extract total RNA including miRNA, miR-Seps3, a candidate for future sepsis diagnosis. The method utilized 100 ul of healthy human blood plasma for manual and robotic extraction method as input volume followed by two-tailed RT-qPCR.
No significant difference was found in terms of TAT for manual and robotic extraction while a significant difference was found in terms of HOT between two methods of extraction. No significant difference was observed between spiked manual extractions and spiked robotic extraction on the basis of absolute quantification. Amplification in No-Template control (NTC) compromised the reliability of the qPCR-primers by indicating potential contamination or nonspecific amplification. The results from non-spiked extractions were untrustworthy due to NTC amplification. Both methods were effective in extracting total RNA and no significant difference was observed between two methods. Which concludes that each method can be chosen corresponding to the experimental requirements. qPCR assay requires optimization to deal with the issue of NTC amplification.