A New Specific and Sensitive RT-qPCR Method Based on Splinted 5′ Ligation for the Quantitative Detection of RNA Species Shorter than microRNAs

Recently, we discovered a new family of unusually short RNAs mapping to 5.8S ribosomal RNA (rRNA) and which we named dodecaRNAs (doRNAs), according to the number of core nucleotides (12 nt) their members contain. To confirm these small RNA-sequencing (RNA-Seq) data, validate the existence of the two...

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Main Authors: Marine Lambert, Abderrahim Benmoussa, Patrick Provost
Format: Article
Language:English
Published: MDPI AG 2021-09-01
Series:Non-Coding RNA
Subjects:
Online Access:https://www.mdpi.com/2311-553X/7/3/59
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author Marine Lambert
Abderrahim Benmoussa
Patrick Provost
author_facet Marine Lambert
Abderrahim Benmoussa
Patrick Provost
author_sort Marine Lambert
collection DOAJ
description Recently, we discovered a new family of unusually short RNAs mapping to 5.8S ribosomal RNA (rRNA) and which we named dodecaRNAs (doRNAs), according to the number of core nucleotides (12 nt) their members contain. To confirm these small RNA-sequencing (RNA-Seq) data, validate the existence of the two overly abundant doRNAs—the minimal core 12-nt doRNA sequence and its + 1-nt variant bearing a 5′ Cytosine, C-doRNA—and streamline their analysis, we developed a new specific and sensitive splinted 5′ ligation reverse transcription (RT)-quantitative polymerase chain reaction (qPCR) method. This method is based on a splint-assisted ligation of an adapter to the 5′ end of doRNAs, followed by RT-qPCR amplification and quantitation. Our optimized protocol, which may discriminate between doRNA, C-doRNA, mutated and precursor sequences, can accurately detect as low as 240 copies and is quantitatively linear over a range of 7 logs. This method provides a unique tool to expand and facilitate studies exploring the molecular and cellular biology of RNA species shorter than microRNAs.
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spelling doaj.art-ba44c75b65e047a9a7423ee1d730b8012023-11-22T14:33:24ZengMDPI AGNon-Coding RNA2311-553X2021-09-01735910.3390/ncrna7030059A New Specific and Sensitive RT-qPCR Method Based on Splinted 5′ Ligation for the Quantitative Detection of RNA Species Shorter than microRNAsMarine Lambert0Abderrahim Benmoussa1Patrick Provost2CHU de Québec Research Center/CHUL Pavilion—Université Laval, 2705 Blvd Laurier, Quebec City, QC G1V 4G2, CanadaCHU de Québec Research Center/CHUL Pavilion—Université Laval, 2705 Blvd Laurier, Quebec City, QC G1V 4G2, CanadaCHU de Québec Research Center/CHUL Pavilion—Université Laval, 2705 Blvd Laurier, Quebec City, QC G1V 4G2, CanadaRecently, we discovered a new family of unusually short RNAs mapping to 5.8S ribosomal RNA (rRNA) and which we named dodecaRNAs (doRNAs), according to the number of core nucleotides (12 nt) their members contain. To confirm these small RNA-sequencing (RNA-Seq) data, validate the existence of the two overly abundant doRNAs—the minimal core 12-nt doRNA sequence and its + 1-nt variant bearing a 5′ Cytosine, C-doRNA—and streamline their analysis, we developed a new specific and sensitive splinted 5′ ligation reverse transcription (RT)-quantitative polymerase chain reaction (qPCR) method. This method is based on a splint-assisted ligation of an adapter to the 5′ end of doRNAs, followed by RT-qPCR amplification and quantitation. Our optimized protocol, which may discriminate between doRNA, C-doRNA, mutated and precursor sequences, can accurately detect as low as 240 copies and is quantitatively linear over a range of 7 logs. This method provides a unique tool to expand and facilitate studies exploring the molecular and cellular biology of RNA species shorter than microRNAs.https://www.mdpi.com/2311-553X/7/3/59small RNA5′ ligationRT-qPCRsplintadapterquantification
spellingShingle Marine Lambert
Abderrahim Benmoussa
Patrick Provost
A New Specific and Sensitive RT-qPCR Method Based on Splinted 5′ Ligation for the Quantitative Detection of RNA Species Shorter than microRNAs
Non-Coding RNA
small RNA
5′ ligation
RT-qPCR
splint
adapter
quantification
title A New Specific and Sensitive RT-qPCR Method Based on Splinted 5′ Ligation for the Quantitative Detection of RNA Species Shorter than microRNAs
title_full A New Specific and Sensitive RT-qPCR Method Based on Splinted 5′ Ligation for the Quantitative Detection of RNA Species Shorter than microRNAs
title_fullStr A New Specific and Sensitive RT-qPCR Method Based on Splinted 5′ Ligation for the Quantitative Detection of RNA Species Shorter than microRNAs
title_full_unstemmed A New Specific and Sensitive RT-qPCR Method Based on Splinted 5′ Ligation for the Quantitative Detection of RNA Species Shorter than microRNAs
title_short A New Specific and Sensitive RT-qPCR Method Based on Splinted 5′ Ligation for the Quantitative Detection of RNA Species Shorter than microRNAs
title_sort new specific and sensitive rt qpcr method based on splinted 5 ligation for the quantitative detection of rna species shorter than micrornas
topic small RNA
5′ ligation
RT-qPCR
splint
adapter
quantification
url https://www.mdpi.com/2311-553X/7/3/59
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