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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MDPI AG
2021-09-01
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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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issn | 2311-553X |
language | English |
last_indexed | 2024-03-10T07:21:34Z |
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series | Non-Coding RNA |
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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