SEARCH · PubMed Health

Results for “strand-specific RNA detection”

Explore indexed PubMed citations for clinical trials, systematic reviews and public health research. Read source abstracts and follow each citation to its original PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

4 recordsLinked to original sources

PubMed2026

Monitoring kinetic changes and restriction of influenza A virus RNA species during infection using a Flu-Stranded CRISPR platform.

UNLABELLED: Influenza A virus (IAV) generates three closely related RNA species: viral RNA (vRNA), complementary RNA (cRNA), and messenger RNA (mRNA), whose strand-specific quantification remains limited by sensitivity and quantitative dynamic range, particularly at low RNA abundance. Here, we developed Flu-Stranded CRISPR-Cas12a, a strand-specific detection platform integrating tagged reverse transcription, segment-specific PCR, and Cas12a collateral cleavage to support quantitative analysis of all three RNA species across a broad dynamic range. The assay enables reliable detection down to 102 copies per reaction, extending the lower quantitative boundary relative to both SYBR Green and TaqMan reverse transcription quantitative PCR (RT-qPCR) under matched conditions. Validated in infected cell lines, murine lung tissues, and clinical nasopharyngeal specimens, the platform enabled subtype-discriminating, strand-resolved detection, including samples near or below the quantitative range of SYBR Green RT-qPCR. Using finely resolved infection time-course analyses in NP and NA segments, we identified a reproducible early vRNA decline within the early post-infection phase. This decline was partially attenuated in RIG-I knockout A549 cells, while subsequent vRNA accumulation was enhanced, consistent with a modulatory rather than essential role for RIG-I in early viral RNA dynamics. Subcellular fractionation localized this decline to cytoplasmic incoming genomes. In contrast, importazole-mediated inhibition of nuclear import abolished vRNA recovery without affecting the early decline, indicating that nuclear entry functionally separates early genome reduction from subsequent productive replication. These findings establish Flu-Stranded CRISPR-Cas12a as a strand-resolved framework for monitoring IAV RNA dynamics and reveal an early window of genome vulnerability during cytoplasmic transit that shapes infection outcome. IMPORTANCE: The early fate of incoming influenza virus genomes remains unclear, limiting our understanding of how infection is established or aborted in host cells. We developed Flu-Stranded CRISPR-Cas12a, a strand-specific platform for sensitive and quantitative analysis of influenza viral RNA (vRNA), complementary RNA (cRNA), and messenger RNA (mRNA) across experimental and clinical samples. Using high-resolution time-course analysis, we identified a reproducible early decline in vRNA during the post-entry phase. Our data suggest that this early genome loss arises from multiple processes, with RIG-I acting as a modulatory factor rather than a primary driver. Subcellular fractionation localized this effect to cytoplasmic incoming genomes, whereas importin-β-mediated nuclear entry was required for subsequent vRNA recovery. These findings support a model of an early cytoplasmic phase of genome attrition that is distinct from replication and provide a framework for understanding early influenza RNA kinetics and for guiding strand-resolved diagnostics and antiviral evaluation.

CRISPR-Cas12a↗

PubMed2025

Northern Blotting: Protocols for Radioactive and Nonradioactive Detection of RNA.

Northern blotting is a common technique in RNA biology, allowing to detect and quantify RNAs of interest following separation by gel electrophoresis, transfer to a membrane, and hybridization of specific anti-complementary labelled probes. In this chapter, we describe our protocol for efficient RNA extraction from yeast, separation on agarose gel, and capillary transfer to a membrane. We provide two different methods for strand-specific detection of several types of RNAs using oligonucleotide probes, the first using radioactive 32P-labelled probes, the second based on nonradioactive digoxigenin-labelled probes.

Blotting, Northern↗

PubMed1979

Total synthesis of a tyrosine suppressor transfer RNA gene. XVII. Transcription, in vitro, of the synthetic gene and processing of the primary transcript to transfer RNA.

Primer- and promoter-dependent transcription of the synthesis gene had been studied. Primer-dependent transcription gave, as a major product, an end-to-end transcript which was strand-specific. The transcript was characterized rigorously by two-dimensional separation and analysis of the oligonucleotides formed on digestion with T1-RNase and pancreatic RNase and by nearest neighbor analyses of the oligonucleotides obtained when different alpha-32P-labeled ribonucleoside triphosphates were used as substrates. Minor products accompanying the major transcript were characterized similarly. The major transcript, when treated with an Escherichia coli S-100 extract, was processed to the tRNATyr with correct 5'- and 3'-ends. The nucleolytic cleavages occurring at the 3'-end were characterized. In promoter-dependent transcription, transcription of a restriction fragment containing phi80psu+III gene and the synthetic gene with and without the promoter were compared. Transcription of the synthetic gene was promoter-dependent and strand-specific, the initiation of transcription occurring at the same point as previously found in vivo. Although the synthetic gene contains only 16 base pairs corresponding to the natural sequence following the C-C-A end, processing of the transcript at the 3'-end occurred normally, the endonucleolytic cleavage being followed by exonucleolytic cleavages. The products of promoter-dependent transcription were completely characterized. An examination of the base modifications of the primary transcript during treatment of the latter with E. coli S-100 extract showed couplete modification of uridine to pseudouridine and partial methylation of uridine to ribosylthymine in TpsiCG sequence and partial formation of pseudouridine in the anticodon loop. However, hardly any formation of 2'-O-methylguanosine or of 2-methylthio-6-isopentenyl adenosine could be detected.

Base Sequence↗

PubMed1975

Strand-specific transcription of polyoma virus DNA-early in productive infection and in transformed cells.

The DNA strand origin of nuclear and cytoplasmic polyoma-specific RNA in productively infected mouse cells and in a line of polyoma-transformed hamster cells was determined by hybridization of unlabeled RNA with radioactively labeled separated strands of polyoma DNA. Early in the productive cycle (10 h postinfection) nuclear viral RNA is complementary to only about 40% of the E strand of viral CNA. No RNA complementary to the L strand was detected even when the RNA was first self-annealed to enrich for possible minor species. Early cytoplasmic RNA is complementary to the same 40% of the E strand. Thus, only that part of the poloma genome which codes for early virual messenger RNA appears to be transcribed. Late in infection, nuclear viral RNA is complementary to most or all of the L strand and to at least 60% of the E strand. Late cytoplasmic viral RNA hybridizes to 40 to 45% of the E strand and 50 to 55% of the L strand. The transformed cell nuclear viral RNA is complementary to 60% of the E strand, whereas cytoplasmic RNA is complementary to 40% of the E strand and comprises the same polyoma-specific sequences as are found in RNA early in productive infection. No L strand transcripts could be detected. Thus, in the transformed cells and late in productive infection, viral RNA sequences in the cytoplasm are a specific subset of those in the nucleus.

Cell Line↗