Targeted Gene-Expression Analysis by Genome-Controlled Reverse Transcription-PCR

Stenman, Jakob; Paju, Annukka; Rissanen, O.; Tenkanen, Tuomas; Haglund, Caj; Räsänen, Jari; Salo, Jarmo A.; Stenman, Ulf‐Hǎkan; Orpana, Arto

doi:10.1373/clinchem.2006.073064

Cited by 5 publications

(17 citation statements)

References 27 publications

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“…HT-29, DLD-1 and COLO 205 cells purchased from ATCC (American Type Culture Collection, Rockville, MD, USA) were maintained as described in Stenman et al [16].…”

Section: Cell Culturementioning

confidence: 99%

“…MMP2, MMP7, MMP9, PLAU, PLAUR and MSH2 clones were purchased from the Incyte Genomics IRAL cDNA clone library (Incyte Genomics, Palo Alto, Calif., USA). The sequences of the cDNA clones were confirmed by sequencing as described previously [16]. The cDNA clones were used as templates when generating cRNAs using MEGAscript High Yield Transcription Kit.…”

Section: In Vitro Synthesis Of Crnamentioning

confidence: 99%

“…Total RNA was extracted from cultured cells using the RNeasy Mini Kit (Qiagen, Hilden, Germany) and DNase was treated as described previously [16]. Total RNA was extracted from endoscopic biopsies with the RNeasy Mini Kit and DNase-treated during the extraction on the membrane of the RNeasy column in accordance with the manufacturer's instructions.…”

Section: Rna Extraction From Cultured Cells and Endoscopic Biopsiesmentioning

confidence: 99%

“…RNA extraction from formalin-fixed paraffin-embedded (FFPE) tissue samples RNA was extracted from FFPE tissue sections as described previously [16].…”

Section: Rna Extraction From Cultured Cells and Endoscopic Biopsiesmentioning

confidence: 99%

“…Sequences of the sequence-modifying reverse transcription primers have been described previously [16].…”

Section: Rna Extraction From Cultured Cells and Endoscopic Biopsiesmentioning

confidence: 99%

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Genome‐controlled reverse transcriptase‐polymerase chain reaction for targeted gene‐expression analysis

Stenman

Räsänen²,

Tenkanen³

et al. 2006

Scandinavian Journal of Clinical and Laboratory Investigation

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“…HT-29, DLD-1 and COLO 205 cells purchased from ATCC (American Type Culture Collection, Rockville, MD, USA) were maintained as described in Stenman et al [16].…”

Section: Cell Culturementioning

confidence: 99%

Section: In Vitro Synthesis Of Crnamentioning

confidence: 99%

Section: Rna Extraction From Cultured Cells and Endoscopic Biopsiesmentioning

confidence: 99%

“…RNA extraction from formalin-fixed paraffin-embedded (FFPE) tissue samples RNA was extracted from FFPE tissue sections as described previously [16].…”

Section: Rna Extraction From Cultured Cells and Endoscopic Biopsiesmentioning

confidence: 99%

“…Sequences of the sequence-modifying reverse transcription primers have been described previously [16].…”

Section: Rna Extraction From Cultured Cells and Endoscopic Biopsiesmentioning

confidence: 99%

See 3 more Smart Citations

Genome‐controlled reverse transcriptase‐polymerase chain reaction for targeted gene‐expression analysis

Stenman

Räsänen²,

Tenkanen³

et al. 2006

Scandinavian Journal of Clinical and Laboratory Investigation

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No Tissue Expression of KRAS or BRAF Mutations in 61 Adult Patients Treated for Esophageal Atresia in Early Childhood

Dang

Sistonen

et al. 2017

Eur J Pediatr Surg

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Extendable blocking probe in reverse transcription for analysis of RNA variants with superior selectivity

Ho¹,

Dang²,

Lintula³

et al. 2014

Nucleic Acids Research

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Here we provide the first strategy to use a competitive Extendable Blocking Probe (ExBP) for allele-specific priming with superior selectivity at the stage of reverse transcription. In order to analyze highly similar RNA variants, a reverse-transcriptase primer whose sequence matches a specific variant selectively primes only that variant, whereas mismatch priming to the alternative variant is suppressed by virtue of hybridization and subsequent extension of the perfectly matched ExBP on that alternative variant template to form a cDNA–RNA hybrid. This hybrid will render the alternative RNA template unavailable for mismatch priming initiated by the specific primer in a hot-start protocol of reverse transcription when the temperature decreases to a level where such mismatch priming could occur. The ExBP-based reverse transcription assay detected BRAF and KRAS mutations in at least 1000-fold excess of wild-type RNA and detection was linear over a 4-log dynamic range. This novel strategy not only reveals the presence or absence of rare mutations with an exceptionally high selectivity, but also provides a convenient tool for accurate determination of RNA variants in different settings, such as quantification of allele-specific expression.

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Targeted Gene-Expression Analysis by Genome-Controlled Reverse Transcription-PCR

Cited by 5 publications

References 27 publications

Genome‐controlled reverse transcriptase‐polymerase chain reaction for targeted gene‐expression analysis

Genome‐controlled reverse transcriptase‐polymerase chain reaction for targeted gene‐expression analysis

No Tissue Expression of KRAS or BRAF Mutations in 61 Adult Patients Treated for Esophageal Atresia in Early Childhood

Extendable blocking probe in reverse transcription for analysis of RNA variants with superior selectivity

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