Quantitative Detection The Stability Of The Circulating CircRNAs Using Droplet Digital PCR

Background: Circular RNAs(circ RNAs)is a novel class of noncoding RNAs that form covalently closed circular loops with neither 5?–3? polarities nor polyadenylated tails.This property makes it stable than linear RNAs and promising for noninvasive molecular diagnosis,but also results in long c DNAs being created by RT enzymes.These long c DNAs lead to an overestimation of circ RNA by q PCR.droplet digital PCR(dd PCR),being an end-point PCR,measures the nucleic acids of interest relying on presence or absence of fluorescence,rather than the fluorescence levels during the reaction as q PCR does,which makes it a potential tool to overcome the effect of the rolling RT products on circ RNA quantification.Objective: The present research was used to study the effect of rolling-circle RT on the circ RNA quantification by q PCR,and evaluated the efficacy of digital PCR for quantification of circ RNA as well as the stability of circ RNA in serum and plasmaDesign and methods(1):(1)Blood samples were collected from healthy volunteers followed by two-step centrifugation and RNA extraction.(2)RNA was pooled and mixed with RT reagents.Afterward,the mixture was equalized and given different prolonged RT times followed by PCR,q PCR,and dd PCR respectively.(3)The PCR products were showed on an agarose gel and further confirmed by Sanger sequencing.circ RNA quantification date of q PCR and dd PCR was used for statistical analysisDesign and methods(2):(1)Blood samples were collected from healthy volunteers(2)The clotted blood and EDTA blood was divided into two portions.For the first portion,blood samples were processed immediately,after that the clarified cell-free serum/plasma was left at room temperature for different times followed by RNA extraction.For the second portion,blood samples were left at room temperature for predesigned times,after that,the clotted blood and EDTA blood was subjected to centrifugation and RNA extraction(3)serum/plasma RNA was measured by dd PCR,and the absolute concentration was used for statistical analysis.Result:(1)circ RNA can produce multiple PCR products of different lengths and with the RT incubation time was prolonged,the longer products increased.(2)With the RT incubation time was prolonged,the circ RNA quantification data of RT-q PCR increased,but the date of RT-dd PCR remained unchanged.(3)The concentration of circ RNA remained unchanged up to 24 hours in promptly-centrifuged serum/plasma(4)In delay-centrifuged serum/plasma,the concentration of circ RNA decreased after the clotted blood/EDTA blood was conserved at room temperature for 24 hours.Conclusions: Rolling-circular RT occurs in circ RNA.This phenomenon leads to the creation of multiple PCR products of different lengths but also an overestimation of circ RNA by q PCR.Dd PCR is an effective way to address these biases and has the potential as a circ RNA quantification method.Circ RNA has the potential for biomarker research as it is detectable and stable up to 24 hours in cell-free serum/plasma.However,pre-analytical processing such as the delay in the separation of hemocytes would affect the circ RNA concentration significantly.Thus,blood samples should be centrifuged promptly to reduce the variation originated from the preparation of serum and plasma.