A novel global cDNA amplification technology directly from an RNA template via reverse transcription
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA427335 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR6413180_1.fastq.gz | Fastqsanger.gz | |||
| SRR6413180_2.fastq.gz | Fastqsanger.gz | |||
| SRR6413181_1.fastq.gz | Fastqsanger.gz | |||
| SRR6413181_2.fastq.gz | Fastqsanger.gz | |||
| SRR6413172_1.fastq.gz | Fastqsanger.gz |
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Project description:We developed a method, Reverse Transcription with Random Displacement Amplification (RT-RamDA), to reverse transcribe and amplify cDNA directly from RNA templates using strand displacement and RNA-dependent DNA polymerase activity. This method resulted in a cDNA yield approximately 100-fold higher than conventional RT with high sensitivity, enabling gene expression profiling of low-expression genes with a small amount of RNA, such as that obtained at the single-cell level.
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Project description:Zhao et al. Amplification Table 1 This experiment was designed to determine the effects of template switching (TS) primer and the type of columns used in ds cDNA cleanup on the fidelity of the T7 based RNA linear amplification. BC91 total RNA was amplified with or without TS primer and with two different ds cDNA cleanup protocols. Set of arrays organized by shared biological context, such as organism, tumors types, processes, etc. Keywords: Logical Set
2005-11-04 | GSE3560 | GEO
Project description:The reverse transcriptases (RTs) encoded by mobile group II intron and other non-LTR-retro-elements differ from retroviral RTs in being able to template switch from the 5' end of one template to the 3' end of another without pre-existing complementarity between the donor and acceptor nucleic acids. Here, we used the ability of a thermostable group II intron RT (TGIRT) to template switch directly from synthetic RNA template/DNA primer duplexes having either a blunt end or a 3'-DNA overhang end to establish a complete kinetic framework for the reaction and identify conditions that more efficiently capture acceptor RNAs or DNAs. The rate and amplitude of template switching are optimal from starter duplexes with a single nucleotide 3'-DNA overhang complementary to the 3' nucleotide of the acceptor RNA, suggesting a role for non-templated nucleotide addition of a complementary nucleotide to the 3’ end of cDNAs synthesized from natural templates. Longer 3'-DNA overhangs progressively decrease the rate of template switching, even when complementary to the 3' end of the acceptor template. Although dependent upon only a single base pair between the donor and acceptor, template switching discriminates against mismatches, which coupled with the high processivity of the enzyme, enables the synthesis of full-length DNA copies of acceptor nucleic acids beginning directly at their 3' end. We discuss possible biological functions of the template-switching activity of group II intron and other non-LTR-retroelements RTs, as well as the optimization of this activity for adapter addition in RNA-and DNA-seq.
2019-11-11 | GSE138200 | GEO
Project description:Zhao et al. Amplification Table 1 This experiment was designed to determine the effects of template switching (TS) primer and the type of columns used in ds cDNA cleanup on the fidelity of the T7 based RNA linear amplification. BC91 total RNA was amplified with or without TS primer and with two different ds cDNA cleanup protocols. Set of arrays organized by shared biological context, such as organism, tumors types, processes, etc. Computed
2005-11-03 | E-GEOD-3560 | biostudies-arrayexpress
Project description:Abstract: BACKGROUND: T7 based linear amplification of RNA is used to obtain sufficient antisense RNA for microarray expression profiling. We optimized and systematically evaluated the fidelity and reproducibility of different amplification protocols using total RNA obtained from primary human breast carcinomas and high-density cDNA microarrays. RESULTS: Using an optimized protocol, the average correlation coefficient of gene expression of 11,123 cDNA clones between amplified and unamplified samples is 0.82 (0.85 when a virtual array was created using repeatedly amplified samples to minimize experimental variation). Less than 4% of genes show changes in expression level by 2-fold or greater after amplification compared to unamplified samples. Most changes due to amplification are not systematic both within one tumor sample and between different tumors. Amplification appears to dampen the variation of gene expression for some genes when compared to unamplified poly(A)+ RNA. The reproducibility between repeatedly amplified samples is 0.97 when performed on the same day, but drops to 0.90 when performed weeks apart. The fidelity and reproducibility of amplification is not affected by decreasing the amount of input total RNA in the 0.3-3 micrograms range. Adding template-switching primer, DNA ligase, or column purification of double-stranded cDNA does not improve the fidelity of amplification. The correlation coefficient between amplified and unamplified samples is higher when total RNA is used as template for both experimental and reference RNA amplification. CONCLUSION: T7 based linear amplification reproducibly generates amplified RNA that closely approximates original sample for gene expression profiling using cDNA microarrays. This SuperSeries is composed of the SubSeries listed below.
2006-03-07 | GSE4349 | GEO
Project description:Bacteria defend themselves from viral infection using diverse immune systems, many of which sense and target foreign nucleic acids. Defense-associated reverse transcriptase (DRT) systems provide an intriguing counterpoint to this immune strategy by instead leveraging DNA synthesis, but the identities and functions of their DNA products remain largely unknown. Here we show that DRT2 systems execute an unprecedented immunity mechanism that involves de novo gene synthesis via rolling-circle reverse transcription of a non-coding RNA (ncRNA). Unbiased profiling of RT-associated RNA and DNA ligands in DRT2-expressing cells revealed that reverse transcription generates concatenated cDNA repeats through programmed template jumping on the ncRNA. The presence of phage then triggers second-strand cDNA synthesis, leading to the production of long double-stranded DNA. Remarkably, this DNA product is efficiently transcribed, generating messenger RNAs that encode a stop codon-less, never-ending ORF (neo) whose translation causes potent growth arrest. Phylogenetic analyses and screening of diverse DRT2 homologs further revealed broad conservation of rolling-circle reverse transcription and Neo protein function. Our work highlights an elegant expansion of genome coding potential through RNA-templated gene creation, and challenges conventional paradigms of genetic information encoded along the one-dimensional axis of genomic DNA.
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Project description:We combined electrophoresis and nanopore sequencing to analyze MarathonRT velocity over the heterogeneous sequences and structures of HOTAIR RNA template and reveal that the local sequences and structures of the template have negligible effect to MarathonRT, and MarathonRT can copy the long RNA in a single turnover, which leads to unusually synchronized primer extension at a constant speed of 25 nt/sec. We further demonstrate that ultra-stable RNA structure insertions do not obstruct MarathonRT, suggesting that MarathonRT can immediately disrupt any RNA structures within the template.
2022-06-12 | GSE195979 | GEO
Project description:Template switching by a group II intron reverse transcriptase: biochemical analysis and implications for RNA-seq
| PRJNA575026 | ENA
Project description:The quality of RNA sequencing data relies on specific priming by the primer used for reverse transcription (RT-primer). Non-specific annealing of the RT-primer to the RNA template can generate reads with incorrect cDNA ends and can cause misinterpretation of data (RT mispriming). This kind of artifact in RNA-seq based technologies is underappreciated and currently no adequate tools exist to computationally remove them from published datasets. We show that mispriming can occur with as little as 2 bases of complementarity at the 3' end of the primer followed by intermittent regions of complementarity. We propose an experimental solution to avoid RT-mispriming by performing RNA-seq using thermostable group II intron derived reverse transcriptase (TGIRT-seq).
2018-06-26 | GSE85163 | GEO
Project description:Bacteria defend themselves from viral predation using diverse immune systems, many of which sense and target foreign nucleic acids. Defense-associated reverse transcriptase (DRT) systems provide an intriguing counterpoint to this immune strategy by instead leveraging DNA synthesis, but the identities and functions of their DNA products remain largely unknown. Here we show that DRT2 systems execute an unprecedented immunity mechanism that involves de novo gene synthesis via rolling-circle reverse transcription of a non-coding RNA (ncRNA). Unbiased profiling of RT-associated RNA and DNA ligands uncovered constitutive synthesis of concatenated cDNA repeats through programmed template jumping on the ncRNA. The presence of phage then triggers second-strand cDNA synthesis, leading to the production of long double-stranded DNA. Remarkably, this nascent ‘gene’ is efficiently transcribed, generating messenger RNAs that encode a stop codon-less, never-ending ORF (neo) whose translation causes potent growth arrest. Phylogenetic analyses and screening of diverse DRT2 homologs further revealed broad conservation of rolling-circle reverse transcription and Neo protein function. Our work highlights an elegant expansion of genome coding potential through RNA-templated gene creation, and challenges conventional paradigms of genetic information encoded along the one-dimensional axis of genomic DNA.
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