3 race system for rapid amplification of cdna ends

Manufactured by Thermo Fisher Scientific

Sourced in United States, Germany

The 3' RACE System for Rapid Amplification of cDNA Ends is a tool used for the rapid amplification of the 3' end of cDNA sequences. It provides a method for obtaining the full-length cDNA sequence of a gene of interest by generating a cDNA library and amplifying the 3' end of the transcript.

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18 protocols using 3 race system for rapid amplification of cdna ends

Viral RNA Extraction and Sequencing

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RNA was extracted from organs or cells by using Trizol reagent (Life Technologies) and Direct-zol RNA MiniPrep kit (Zymo Research, Irvine, CA) as previously described [16 (link)]. The reverse transcription was carried out by using the Superscript III First-Strand Synthesis System (Life Technologies) and random primers according to manufacturer's protocol. cDNAs were amplified by PCR into two and three DNA fragments for viral S and L segments, respectively. The PCR products were purified using a QIAquick PCR purification kit (Qiagen) and directly sequenced using an ABI Prism 3130xl DNA sequencer (Life Technologies). To determine the 5’ and 3’ UTR of the S and L segments, 5’ RACE System for Rapid Amplification of cDNA Ends and the 3’ RACE System for Rapid Amplification of cDNA Ends were used, respectively (Life Technologies).

Koma T., Huang C., Aronson J.F., Walker A.G., Miller M., Smith J.N., Patterson M, & Paessler S. (2016). The Ectodomain of Glycoprotein from the Candid#1 Vaccine Strain of Junin Virus Rendered Machupo Virus Partially Attenuated in Mice Lacking IFN-αβ/γ Receptor. PLoS Neglected Tropical Diseases, 10(8), e0004969.

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Rapid Amplification of cDNA Ends (RACE) Protocol

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3’-RACE was performed using the 3’-RACE System for Rapid Amplification of cDNA ends (Life Technologies), according to the manufacturer’s protocol, in 20 μl reactions containing 200 ng of total RNA template. 5’-RACE was performed using the 5’-RACE System for Rapid Amplification of cDNA ends, Version 2.0 (Life Technologies), according to the manufacturer’s protocol, in 25 μl reactions containing 500 ng of total RNA template and 2.5 pmol of gene-specific primer. Primary and nested PCR reactions were performed using Platinum Taq polymerase (Life Technologies), according to the manufacturer’s protocol, in 50 μl reactions containing 500 nM gene-specific primer. A list of primers used in the study is provided in S1 Fig. PCR products were cloned using the TOPO TA Cloning Kit (Life Technologies) according to the manufacturer’s protocol. Individual colonies were grown in L-Broth, and plasmids were purified using the PureYield Plasmid Miniprep System (Promega) and then sequenced (Source BioScience).

Hector R.D., Dando O., Landsberger N., Kilstrup-Nielsen C., Kind P.C., Bailey M.E, & Cobb S.R. (2016). Characterisation of CDKL5 Transcript Isoforms in Human and Mouse. PLoS ONE, 11(6), e0157758.

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Viral RNA Extraction and Sequencing

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RNAs were extracted from homogenized organs or lysed cell lines by using Trizol reagent (Life Technologies) and Direct-zol RNA MiniPrep kits (Zymo Research, Irvine, CA) as previously described [36 (link)]. Reverse transcription was performed by using the Superscript III First-Strand Synthesis System (Life Technologies) with random primers according to the manufacturer’s protocol. cDNAs for GPC ORF were amplified by PCR with 5’-CGCACCGGGGATCCTAGGCGATTC-3’ and 5’-CCTCTCAGCCTTCTATTTCTACCC-3’. cDNAs for whole virus genome were amplified by PCR into two and three DNA fragments for viral S with previously described primers [37 (link)] and L segments with the following primers, respectively: 5’- CGCACCGGGGATCCTAGGCGTAAC-3’ and 5’-TAGGAACTGTGCCAGAAAGG-3’, 5’-TCTCTAACGCACTTGCTACC-3’ and 5’-TTGGATGTGCTGTGGTGAAC-3’, 5’-GAGGATGTTGCCTAACTC-3’ and 5’-CGCACCGAGGATCCTAGGCGACAC-3’. To read the sequence of the 5’ and 3’ end, the 5’ RACE System for Rapid Amplification of cDNA Ends (Life Technologies) and the 3’ RACE System for Rapid Amplification of cDNA Ends (Life Technologies) were used according to the manufacturer’s protocol. After purification using a QIAquick PCR purification kit (Qiagen), PCR products were directly sequenced using an ABI Prism 3130xl DNA sequencer (Life Technologies).

Koma T., Huang C., Coscia A., Hallam S., Manning J.T., Maruyama J., Walker A.G., Miller M., Smith J.N., Patterson M., Abraham J, & Paessler S. (2021). Glycoprotein N-linked glycans play a critical role in arenavirus pathogenicity. PLoS Pathogens, 17(3), e1009356.

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3'RACE for Ribosomal and mRNA Detection

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Total RNA was treated with poly(A) polymerase (NEB) following the supplier's recommendations. 3′-RACE was performed using the 3′ RACE System for Rapid Amplification of cDNA Ends (Thermofisher). Total poly(A) RNA was reverse transcribed and used for PCR amplification of the cDNA molecules that correspond to the 23S, 16S or 5S rRNAs and tmRNA using the primers described in Supplementary Table S2 and an oligo dT supplied with the kit. The resulting fragments were cloned into the pGEM-T Easy system (Promega) and blue/white colony screening was performed on LB plates containing 100 μg/ml ampicillin, 1 mM IPTG and 20 μg/ml X-Gal (5-bromo-4-chloro-3-indolyl-ß-d-galactopyranoside). The fragments cloned in the pGEM-T multicloning site of 10 individual white colonies for each gene of interest were PCR amplified and sequenced (Eurofins Genomics).

Tejada-Arranz A., Matos R.G., Quentin Y., Bouilloux-Lafont M., Galtier E., Briolat V., Kornobis E., Douché T., Matondo M., Arraiano C.M., Raynal B, & De Reuse H. (2021). RNase R is associated in a functional complex with the RhpA DEAD-box RNA helicase in Helicobacter pylori. Nucleic Acids Research, 49(9), 5249-5264.

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Transcriptome analysis of sfpq knockout

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RNA was extracted from 24 hpf sfpq^−/− embryos using the RNeasy Mini Kit (Qiagen). Reverse transcription was performed using the 3′ RACE System for Rapid Amplification of cDNA Ends (ThermoFisher). cDNA was amplified in two subsequent PCR reactions, using the adapter primer as a reverse primer and the primers listed in Supplementary Data 9 as forward primers. After the second amplification step, PCR products were gel-purified and sequenced directly.

Gordon P.M., Hamid F., Makeyev E.V, & Houart C. (2021). A conserved role for the ALS-linked splicing factor SFPQ in repression of pathogenic cryptic last exons. Nature Communications, 12, 1918.

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RACE PCR for PyroLuc Validation

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RACE PCR was used to validate the presence of our PyroLuc in another Canadian sample. Specifically, we performed 3′ RACE System for Rapid Amplification of cDNA Ends (ThermoFisher# 18373-019) and 5′ RACE System for Rapid Amplification of cDNA Ends (ThermoFisher# 18374-058).

Tessler M., Gaffney J.P., Oliveira A.G., Guarnaccia A., Dobi K.C., Gujarati N.A., Galbraith M., Mirza J.D., Sparks J.S., Pieribone V.A., Wood R.J, & Gruber D.F. (2020). A putative chordate luciferase from a cosmopolitan tunicate indicates convergent bioluminescence evolution across phyla. Scientific Reports, 10, 17724.

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3' RACE Experiments for AaCOOLAIR

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3′ RACE experiments were performed on 3 μg of total RNA with the 3′ RACE System for Rapid amplification of cDNA ends (Invitrogen) according to the manufacturer’s protocol. PCR followed by a nested PCR were performed using AaCOOLAIR-specific forward primers (see Supplementary Table 3) together with the provided Universal Amplification Primer and the Abridge Universal Amplification Primer respectively. PCR products were cloned and sequenced.

Castaings L., Bergonzi S., Albani M.C., Kemi U., Savolainen O, & Coupland G. (2014). Evolutionary conservation of cold-induced antisense RNAs of FLOWERING LOCUS C in Arabidopsis thaliana perennial relatives. Nature Communications, 5, 4457.

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Cloning and Validation of Plasmid Constructs

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pLKO.1, pCDH, and pCMV were used in our study. ShRNA sequences were generated by PCR and then cloned into the pLKO.1 vector. The KRT80 and lncHNSCAT1 overexpression cassette was generated by PCR, cloned into the pCDH vector, and verified by DNA sequencing. A rapid amplification of cDNA ends (RACE) assay was carried out with the 5′ RACE System for Rapid Amplification of cDNA Ends (Invitrogen, #18374-058) and the 3′ RACE System for Rapid Amplification of cDNA Ends (Invitrogen, #18373-019) according to the manufacturer's protocols. The amplified DNA fragment was cloned into the pGEM-T Easy vector and validated by Sanger sequencing (Sangon Biotech, China).

Zhao Y., Huang X., Zhang Z., Li H, & Zan T. (2022). The Long Noncoding Transcript HNSCAT1 Activates KRT80 and Triggers Therapeutic Efficacy in Head and Neck Squamous Cell Carcinoma. Oxidative Medicine and Cellular Longevity, 2022, 4156966.

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Rapid Amplification of cDNA Ends (RACE)

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Rapid amplification of cDNA ends (RACE) assays were carried out with the 5′ RACE System for Rapid Amplification of cDNA Ends, Version 2.0 (Invitrogen, #18374-058) and the 3′ RACE System for Rapid Amplification of cDNA Ends (Invitrogen, #18373-019), according to the manufacturer’s protocols. For 5′-RACE, 5 μg of tobacco acid pyrophosphatase (Epicentre Technologies; #RP8092H) treated RNA was reverse transcribed using a sRNA-specific antisense primer and SuperScript^TM Reverse Transcriptase Invitrogen; #18091050). cDNA was then purified, dC-tailed, and used as a template in a PCR reaction with the Abridged Anchor Primer (AAP) and a nested gene-specific primer. 3′-RACE was performed by ligating a poly(A) tail, using a Poly(A) Polymerase Tailing Kit (Epicentre; #PAP5104H) before reverse transcription. Specific cDNAs were then directly amplified by PCR using an Anchor Primer (AP) that targets the poly(A) tail region and a gene-specific primer that anneals to a region of known sRNA sequence. PCR products were cloned into the pGEM^®-T Vector System (Promega; #A3600) before sequencing. Primers used in RACE assays are presented in Supplementary Table S2.

Han R., Xu L., Wang T., Liu B, & Wang L. (2017). A Small Regulatory RNA Contributes to the Preferential Colonization of Escherichia coli O157:H7 in the Large Intestine in Response to a Low DNA Concentration. Frontiers in Microbiology, 8, 274.

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CEACAM32 mRNA 3' End Amplification

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For the amplification of the 3′ end of CEACAM32 mRNA we used the 3´ RACE System for Rapid Amplification of cDNA Ends from Invitrogen according to the standard protocol. The amplicons were isolated from an agarose gel using the QIAquick Gel Extraction Kit from Qiagen and sequenced using the BigDye Terminator Cycle Sequencing Kit (PE Applied Biosystems). This sequence was used to design specific primers to amplify full-length CEACAM32 cDNA. The sequences of the PCR products were determined by direct nucleotide sequencing.

Hänske J., Hammacher T., Grenkowitz F., Mansfeld M., Dau T.H., Maksimov P., Friedrich C., Zimmermann W, & Kammerer R. (2020). Natural selection supports escape from concerted evolution of a recently duplicated CEACAM1 paralog in the ruminant CEA gene family. Scientific Reports, 10, 3404.

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