Using bisulfite conversion of RNA with RT‐PCR to unambiguously quantify expression of overlapping sense and antisense RNA in skeletal muscle
Authors: Theodore Lindsey A. , Gilbert Carolyn A. , Pandorf Clay E. 
Published: November 2023

Long non‐coding RNAs (lncRNAs) have been revealed as being regulators of transcription of protein‐coding genes and may play critical roles in the remodeling of skeletal muscle in response to inactivity. We investigated lncRNAs that are transcribed antisense to and overlapping Myh7 (Type I Myosin Heavy Chain). We sought to develop a new RT‐PCR based approach to quantify overlapping sense (S) and antisense (AS) RNA that avoids amplification of non‐specific cDNA (NSC) products. Reverse transcription–polymerase chain reaction (RT‐PCR) is the go‐to method for quantitating RNA transcripts, due in part to its high sensitivity. Quantifying S‐AS gene pairs by RT‐PCR requires gene specific primers to be used in the RT. However, NSC is often transcribed, particularly when overlapping, and thus complementary transcripts are present. NSC is defined as PCR product produced from an RT in which primers are omitted from the RT reaction. On the other hand, gene‐specific cDNA (GSC) is defined as PCR product produced from gene specific primers in the RT reaction. NSC products that amplify during PCR may interfere with accurate quantification of the intended target. We developed an alternative method that utilizes the advantages of RT‐PCR without the specificity issues. We utilized bisulfite conversion of RNA, which converts all cytidine residues to uridine residues, thus rendering overlapping RNA to become non‐complementary and allowing for the unambiguous quantification of S and AS strands when quantified by RT‐PCR. Utilizing this approach with DNase‐treated RNA from Soleus (SOL) muscle of hindlimb suspended (HS) and normal ambulatory (CON) rats (N=6) we examined sense and antisense transcripts in the Myh7 coding region (at +6kb) and upstream from Myh7 at −9kb from the transcription start site. We compared bisulfite converted (BC) and non‐converted (NC) RNA. Data was normalized to beta‐actin mRNA.Using endpoint PCR to detect AS RNA in the coding region of Myh7 we determined that levels of NSC were 66% of the abundance of GSC. Such a high level of cDNA “background” for the AS target may compromise the validity of the quantitation. In contrast, cDNA derived from BC RNA revealed that NSC was undetectable when targeting both the S and the AS transcripts. However, in comparing differential expression of AS Myh7 RNA in HS as compared to CON both BC and NC RNA had the same result; there was not a significant difference. Examination S RNA at −9kb to Myh7 showed that levels of NSC averaged 71% of the abundance of GSC derived from NC RNA. NSC was again undetectable in the BC RNA. Differential expression of S RNA at −9kb in HS as compared to CON was 1.3‐fold (non‐significant) as assessed from NC RNA, and 0.81‐fold (non‐significant) as quantified in BC RNA. Though a significant HS vs CON difference was not apparent with either RNA treatments, the direction of change was not the same in NC as compared to BC RNA. In conclusion, this analysis showed that bisulfite conversion of RNA followed by RT‐PCR yields cDNA free of non‐specific products that may otherwise preclude accurate quantification of lncRNA expression in response to treatments such as altered loading states in muscle.