Novel 5′/3′RACE Method for Amplification and Determination of Single-Stranded RNAs Through Double-Stranded RNA (dsRNA) Intermediates

P. Pankovics, Ákos Boros, G. Reuter

Research output: Article

1 Citation (Scopus)

Abstract

To acquire the full-length sequences and to determine the 5′/3′ends of the RNA genomes and mRNA transcripts using the rapid amplification of cDNA ends (RACE) protocols—via cDNA or mRNA templates—are a great challenge. This 4-steps RNA-based RACE method uses different ways to determine the RNA ends through a double-stranded (ds) RNA intermediate (dsRNA-RACE). In the first step a complementary RNA strand is synthesised by Phi6 RNA replicase enzyme next to the template ssRNA forming a dsRNA intermediate. The following steps include adapter ligation, nucleic acid purification and two classical methods with minor modifications reverse transcription and polymerase chain reaction. The dsRNA-RACE protocol could be used in wide variety of ssRNA (cellular, viral, bacterial, etc.) templates in the field of microbiology and cellular biology and suitable for the amplification of full-length RNAs including the 5′/3′ends. This is a novel, expansively utilizable molecular tool with fewer disadvantages than the existing 5′/3′RACE approaches.

Original languageEnglish
Pages (from-to)974-981
Number of pages8
JournalMolecular Biotechnology
Volume57
Issue number11-12
DOIs
Publication statusPublished - dec. 1 2015

Fingerprint

Double-Stranded RNA
RNA
Amplification
Complementary DNA
Cytology
RNA Replicase
Microbiology
Complementary RNA
Messenger RNA
Polymerase chain reaction
Transcription
Nucleic Acids
Reverse Transcription
Purification
Ligation
Cell Biology
Genes
Genome
Polymerase Chain Reaction
Nucleic acids

ASJC Scopus subject areas

  • Biochemistry
  • Biotechnology
  • Molecular Biology
  • Bioengineering
  • Applied Microbiology and Biotechnology

Cite this

@article{be9fc145e25c4cdeb356f241b03b1235,
title = "Novel 5′/3′RACE Method for Amplification and Determination of Single-Stranded RNAs Through Double-Stranded RNA (dsRNA) Intermediates",
abstract = "To acquire the full-length sequences and to determine the 5′/3′ends of the RNA genomes and mRNA transcripts using the rapid amplification of cDNA ends (RACE) protocols—via cDNA or mRNA templates—are a great challenge. This 4-steps RNA-based RACE method uses different ways to determine the RNA ends through a double-stranded (ds) RNA intermediate (dsRNA-RACE). In the first step a complementary RNA strand is synthesised by Phi6 RNA replicase enzyme next to the template ssRNA forming a dsRNA intermediate. The following steps include adapter ligation, nucleic acid purification and two classical methods with minor modifications reverse transcription and polymerase chain reaction. The dsRNA-RACE protocol could be used in wide variety of ssRNA (cellular, viral, bacterial, etc.) templates in the field of microbiology and cellular biology and suitable for the amplification of full-length RNAs including the 5′/3′ends. This is a novel, expansively utilizable molecular tool with fewer disadvantages than the existing 5′/3′RACE approaches.",
keywords = "5′/3′RACE, mRNA, PCR, RNA genome, ssRNA",
author = "P. Pankovics and {\'A}kos Boros and G. Reuter",
year = "2015",
month = "12",
day = "1",
doi = "10.1007/s12033-015-9889-7",
language = "English",
volume = "57",
pages = "974--981",
journal = "Molecular Biotechnology",
issn = "1073-6085",
publisher = "Humana Press",
number = "11-12",

}

TY - JOUR

T1 - Novel 5′/3′RACE Method for Amplification and Determination of Single-Stranded RNAs Through Double-Stranded RNA (dsRNA) Intermediates

AU - Pankovics, P.

AU - Boros, Ákos

AU - Reuter, G.

PY - 2015/12/1

Y1 - 2015/12/1

N2 - To acquire the full-length sequences and to determine the 5′/3′ends of the RNA genomes and mRNA transcripts using the rapid amplification of cDNA ends (RACE) protocols—via cDNA or mRNA templates—are a great challenge. This 4-steps RNA-based RACE method uses different ways to determine the RNA ends through a double-stranded (ds) RNA intermediate (dsRNA-RACE). In the first step a complementary RNA strand is synthesised by Phi6 RNA replicase enzyme next to the template ssRNA forming a dsRNA intermediate. The following steps include adapter ligation, nucleic acid purification and two classical methods with minor modifications reverse transcription and polymerase chain reaction. The dsRNA-RACE protocol could be used in wide variety of ssRNA (cellular, viral, bacterial, etc.) templates in the field of microbiology and cellular biology and suitable for the amplification of full-length RNAs including the 5′/3′ends. This is a novel, expansively utilizable molecular tool with fewer disadvantages than the existing 5′/3′RACE approaches.

AB - To acquire the full-length sequences and to determine the 5′/3′ends of the RNA genomes and mRNA transcripts using the rapid amplification of cDNA ends (RACE) protocols—via cDNA or mRNA templates—are a great challenge. This 4-steps RNA-based RACE method uses different ways to determine the RNA ends through a double-stranded (ds) RNA intermediate (dsRNA-RACE). In the first step a complementary RNA strand is synthesised by Phi6 RNA replicase enzyme next to the template ssRNA forming a dsRNA intermediate. The following steps include adapter ligation, nucleic acid purification and two classical methods with minor modifications reverse transcription and polymerase chain reaction. The dsRNA-RACE protocol could be used in wide variety of ssRNA (cellular, viral, bacterial, etc.) templates in the field of microbiology and cellular biology and suitable for the amplification of full-length RNAs including the 5′/3′ends. This is a novel, expansively utilizable molecular tool with fewer disadvantages than the existing 5′/3′RACE approaches.

KW - 5′/3′RACE

KW - mRNA

KW - PCR

KW - RNA genome

KW - ssRNA

UR - http://www.scopus.com/inward/record.url?scp=84945445126&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=84945445126&partnerID=8YFLogxK

U2 - 10.1007/s12033-015-9889-7

DO - 10.1007/s12033-015-9889-7

M3 - Article

C2 - 26315976

AN - SCOPUS:84945445126

VL - 57

SP - 974

EP - 981

JO - Molecular Biotechnology

JF - Molecular Biotechnology

SN - 1073-6085

IS - 11-12

ER -