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Here, we sought to determine the discriminatory capacity between these two closely-related miRNA isoforms of three alternative poly(A) based-RT-qPCR strategies in both synthetic and real biological context. We found that these poly(A) RT-qPCR strategies exhibit a significant cross-reactivity between these miR-31-5p isomiRs which differ by a single nucleotide, compromising the reliable quantification of individual miRNA isoforms. Fortunately, in the biological context, given that the two miRNA variants show changes in the same direction, RT-qPCR results were consistent with the findings of small RNA-seq study. We suggest that miRNA selection for RT-qPCR validation should be performed with care, prioritizing those canonical miRNAs that, in small RNA-seq, show parallel/homogeneous expression behavior with their most prevalent isomiRs, to avoid confounding RT-qPCR-based results. This is suggested as the current best strategy for robust biomarker selection to develop clinically useful tests. Keywords: miRNA isoforms; isomiRs; poly(A)-RT-qPCR; selective amplification; small RNAseq validation strategy Author: Adriana Ferre, Lucía Santiago, José Francisco Sánchez-Herrero, Olga López-Rodrigo, Josvany Sánchez-Curbelo, Lauro Sumoy, Lluís Bassas and Sara Larriba Creator: LaTeX with hyperref Producer: pdfTeX-1.40.21 CreationDate: Thu Oct 26 08:44:30 2023 CEST ModDate: Thu Oct 26 09:00:00 2023 CEST Custom Metadata: no Metadata Stream: no Tagged: no UserProperties: no Suspects: no Form: none JavaScript: no Pages: 14 Encrypted: no Page size: 595.276 x 841.89 pts (A4) Page rot: 0 File size: 1091267 bytes Optimized: no PDF version: 1.7 name type encoding emb sub uni object ID ------------------------------------ ----------------- ---------------- --- --- --- --------- JYPLJJ+VnURWPalladioL-Bold Type 1 Custom yes yes no 10 0 HJJCXD+URWPalladioL-Bold Type 1 Custom yes yes no 15 0 VOAGQP+URWPalladioL-Roma Type 1 Custom yes yes no 20 0 YFFKLH+CMSY10 Type 1 Builtin yes yes no 24 0 NYFOCY+URWPalladioL-Ital Type 1 Custom yes yes no 28 0 PXEKZD+CMBSY10 Type 1 Builtin yes yes no 32 0 IRWHZX+TimesNewRomanPSMT Type 1C WinAnsi yes yes no 50 0 CKUPDE+TimesNewRomanPS-BoldItalicMT Type 1C WinAnsi yes yes no 53 0 BCDEEE+PalatinoLinotype-Bold TrueType WinAnsi yes yes no 86 0 TimesNewRomanPS-BoldMT TrueType WinAnsi no no no 90 0 BCDFEE+PalatinoLinotype-Roman TrueType WinAnsi yes yes no 93 0 BCDGEE+PalatinoLinotype-Italic TrueType WinAnsi yes yes no 97 0 BCDHEE+PalatinoLinotype-Italic CID TrueType Identity-H yes yes yes 101 0 BCDIEE+PalatinoLinotype-Bold CID TrueType Identity-H yes yes yes 108 0 BCDJEE+PalatinoLinotype-Roman CID TrueType Identity-H yes yes yes 115 0 BCDEEE+PalatinoLinotype-Bold TrueType WinAnsi yes yes no 138 0 BCDFEE+PalatinoLinotype-Roman TrueType WinAnsi yes yes no 142 0 BCDGEE+PalatinoLinotype-Italic TrueType WinAnsi yes yes no 146 0 BCDHEE+PalatinoLinotype-Italic CID TrueType Identity-H yes yes yes 150 0 BCDIEE+PalatinoLinotype-Bold CID TrueType Identity-H yes yes yes 157 0 BCDJEE+PalatinoLinotype-Roman CID TrueType Identity-H yes yes yes 164 0 OPZDHV+VnURWPalladioL Type 1 Custom yes yes no 181 0 URYLRZ+URWPalladioL-BoldItal Type 1 Custom yes yes no 188 0 OVNEXW+EURM10 Type 1 Builtin yes yes no 192 0 Jhove (Rel. 1.28.0, 2023-05-18) Date: 2024-09-07 02:53:33 CEST RepresentationInformation: 300101.pdf ReportingModule: PDF-hul, Rel. 1.12.4 (2023-03-16) LastModified: 2024-09-06 12:52:22 CEST Size: 1091267 Format: PDF Version: 1.7 Status: Well-Formed and valid SignatureMatches: PDF-hul MIMEtype: application/pdf PDFMetadata: Objects: 353 FreeObjects: 1 IncrementalUpdates: 0 DocumentCatalog: PageLayout: SinglePage PageMode: UseNone Outlines: Item: Title: Introduction Destination: section.1 Item: Title: Results Destination: section.2 Children: Item: Title: Assaying One Synthetic IsomiR at a Time Destination: subsection.2.1 Item: Title: Assaying the Two Synthetic IsomiRs Simultaneously Destination: subsection.2.2 Item: Title: Assaying the Two IsomiRs in Biological Samples Destination: subsection.2.3 Item: Title: Discussion Destination: section.3 Item: Title: Materials and Methods Destination: section.4 Children: Item: Title: Subjects of Study Destination: subsection.4.1 Item: Title: Sample Collection and Processing Destination: subsection.4.2 Item: Title: Isolation of Small EVs from Seminal Plasma Destination: subsection.4.3 Item: Title: Small RNA-Containing Total RNA Isolation Destination: subsection.4.4 Item: Title: sncRNA Quantification by RT-qPCR Destination: subsection.4.5 Children: Item: Title: miRCURY LNA miRNA PCR System (Qiagen) Destination: subsubsection.4.5.1 Item: Title: miRPrimer2 Strategy Destination: subsubsection.4.5.2 Item: Title: Modified miR-X Poly(A) RT-qPCR Protocol Destination: subsubsection.4.5.3 Item: Title: Statistical Analysis Destination: subsection.4.6 Item: Title: References Destination: section.A. Info: Title: 3'IsomiR Species Composition Affects Reliable Quantification of miRNA/isomiR Variants by Poly(A) RT-qPCR: Impact on Small RNA-Seq Profiling Validation Author: Adriana Ferre, Lucía Santiago, José Francisco Sánchez-Herrero, Olga López-Rodrigo, Josvany Sánchez-Curbelo, Lauro Sumoy, Lluís Bassas and Sara Larriba Subject: Small RNA-sequencing (small RNA-seq) has revealed the presence of small RNA-naturally occurring variants such as microRNA (miRNA) isoforms or isomiRs. Due to their small size and the sequence similarity among miRNA isoforms, their validation by RT-qPCR is challenging. We previously identified two miR-31-5p isomiRs the canonical and a 3'isomiR variant (3' G addition) which were differentially expressed between individuals with azoospermia of different origin. Here, we sought to determine the discriminatory capacity between these two closely-related miRNA isoforms of three alternative poly(A) based-RT-qPCR strategies in both synthetic and real biological context. We found that these poly(A) RT-qPCR strategies exhibit a significant cross-reactivity between these miR-31-5p isomiRs which differ by a single nucleotide, compromising the reliable quantification of individual miRNA isoforms. Fortunately, in the biological context, given that the two miRNA variants show changes in the same direction, RT-qPCR results were consistent with the findings of small RNA-seq study. We suggest that miRNA selection for RT-qPCR validation should be performed with care, prioritizing those canonical miRNAs that, in small RNA-seq, show parallel/homogeneous expression behavior with their most prevalent isomiRs, to avoid confounding RT-qPCR-based results. This is suggested as the current best strategy for robust biomarker selection to develop clinically useful tests. 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