Sequencing of cDNA using anchored oligo dT primers

Thomas, Mark G.; Hesse, Sarah A.; McKie, Andrew T.; Farzaneh, Farzin

doi:10.1093/nar/21.16.3915

Cited by 8 publications

(4 citation statements)

References 4 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…We observed that oligonucleotide hybridization with gmRNA masks the binding site of the anchored oligo-T primer (T 30 VN, oligonucleotides are shown in Supplementary Table 1 ), suppressing gcDNA synthesis. The anchored oligo-T primer has degenerated nucleotide(s) at 3′ end to determine cDNA priming at single position per transcript 9 10 . Non-anchored oligo-T primers anneal randomly along 150–250 poly-A nucleotide tail of mRNA 11 , producing multiple cDNA molecules from a single template mediated by strand replacement activity of reverse transcriptase (RT).…”

Section: Resultsmentioning

confidence: 99%

Globin mRNA reduction for whole-blood transcriptome sequencing

Krjutškov

Koel

Roost

et al. 2016

Sci Rep

View full text Add to dashboard Cite

The transcriptome analysis of whole-blood RNA by sequencing holds promise for the identification and tracking of biomarkers; however, the high globin mRNA (gmRNA) content of erythrocytes hampers whole-blood and buffy coat analyses. We introduce a novel gmRNA locking assay (GlobinLock, GL) as a robust and simple gmRNA reduction tool to preserve RNA quality, save time and cost. GL consists of a pair of gmRNA-specific oligonucleotides in RNA initial denaturation buffer that is effective immediately after RNA denaturation and adds only ten minutes of incubation to the whole cDNA synthesis procedure when compared to non-blood RNA analysis. We show that GL is fully effective not only for human samples but also for mouse and rat, and so far incompletely studied cow, dog and zebrafish.

show abstract

Section: Resultsmentioning

confidence: 99%

Globin mRNA reduction for whole-blood transcriptome sequencing

Krjutškov

Koel

Roost

et al. 2016

Sci Rep

View full text Add to dashboard Cite

show abstract

“…Anchor bases were added to fix the primers at the immediate base following the polynucleotide tail (Thomas et al, 1993). The use of a RT primer without an anchor could promote base pairing of the RT primer at different positions in the added polynucleotide tail, thus resulting in PCR products with a different number of added nucleotides, which might possibly blur the sequences obtained at the final step.…”

Section: Discussionmentioning

confidence: 99%

An alternative method to determine the 5′ extremities of non-segmented, negative sense RNA viral genomes using positive replication intermediate 3′ tailing: Application to two members of the Paramyxoviridae family

Brown

Briand

Guionie

et al. 2013

Journal of Virological Methods

View full text Add to dashboard Cite

“…Total RNA was chemically fragmented using DNA Fragmentation Reagents (Ambion) for 5 min at 70°C in 10 mL, LiCl precipitated in the presence of glycogen and resuspended in water. At room temperature, 1 mL of BPM1polyT22 primer (100 mM) (Biotin-GGCCAG TCCTGGAGTTTTTTTTTTTTTTTTTTTTTTVN) with a 39 anchor sequence (Thomas et al 1993) was annealed to 200 mg of streptavidin magnetic beads (NEB), and the beads were washed in binding buffer (0.5 M NaCl, 20 mM Tris-HCl at pH 7.5, 1 mM EDTA). RNA in 100 mL of 13 binding buffer was annealed to the primer for 30 min at room temperature, washed three times in 13 binding buffer and once in low-salt buffer (0.15 M NaCl, 20 mM Tris-HCl at pH 7.5, 1 mM EDTA).…”

Section: -End Prediction and Modificationmentioning

confidence: 99%

Incorporating RNA-seq data into the zebrafish Ensembl genebuild

et al. 2012

View full text Add to dashboard Cite

Ensembl gene annotation provides a comprehensive catalog of transcripts aligned to the reference sequence. It relies on publicly available species-specific and orthologous transcripts plus their inferred protein sequence. The accuracy of gene models is improved by increasing the species-specific component that can be cost-effectively achieved using RNA-seq. Two zebrafish gene annotations are presented in Ensembl version 62 built on the Zv9 reference sequence. Firstly, RNAseq data from five tissues and seven developmental stages were assembled into 25,748 gene models. A 39-end capture and sequencing protocol was developed to predict the 39 ends of transcripts, and 46.1% of the original models were subsequently refined. Secondly, a standard Ensembl genebuild, incorporating carefully filtered elements from the RNA-seqonly build, followed by a merge with the manually curated VEGA database, produced a comprehensive annotation of 26,152 genes represented by 51,569 transcripts. The RNA-seq-only and the Ensembl/VEGA genebuilds contribute contrasting elements to the final genebuild. The RNA-seq genebuild was used to adjust intron/exon boundaries of orthologous defined models, confirm their expression, and improve 39 untranslated regions. Importantly, the inferred protein alignments within the Ensembl genebuild conferred proof of model contiguity for the RNA-seq models. The zebrafish gene annotation has been enhanced by the incorporation of RNA-seq data and the pipeline will be used for other organisms. Organisms with little species-specific cDNA data will generally benefit the most.

show abstract

Sequencing of cDNA using anchored oligo dT primers

Cited by 8 publications

References 4 publications

Globin mRNA reduction for whole-blood transcriptome sequencing

Globin mRNA reduction for whole-blood transcriptome sequencing

An alternative method to determine the 5′ extremities of non-segmented, negative sense RNA viral genomes using positive replication intermediate 3′ tailing: Application to two members of the Paramyxoviridae family

Incorporating RNA-seq data into the zebrafish Ensembl genebuild

Contact Info

Product

Resources

About