Since its introduction, the 'single-step' method has become widely used for isolating total RNA from biological samples of different sources. The principle at the basis of the method is that RNA is separated from DNA after extraction with an acidic solution containing guanidinium thiocyanate, sodium acetate, phenol and chloroform, followed by centrifugation. Under acidic conditions, total RNA remains in the upper aqueous phase, while most of DNA and proteins remain either in the interphase or in the lower organic phase. Total RNA is then recovered by precipitation with isopropanol and can be used for several applications. The original protocol, enabling the isolation of RNA from cells and tissues in less than 4 hours, greatly advanced the analysis of gene expression in plant and animal models as well as in pathological samples, as demonstrated by the overwhelming number of citations the paper gained over 20 years.

The single-step method of RNA isolation by acid guanidinium thiocyanate–phenol–chloroform extraction: twenty-something years on

Reverse transcription PCR (RT-PCR) represents a sensitive and powerful tool for analyzing RNA. While it has tremendous potential for quantitative applications, a comprehensive knowledge of its technical aspects is required. Successful quantitative RT-PCR involves correction for experimental variations in individual RT and PCR efficiencies. This review addresses the mathematics of RT-PCR, choice of RNA standards (internal vs. external) and quantification strategies (competitive, noncompetitive and kinetic [real-time] amplification). Finally, the discussion turns to practical considerations in experimental design. It is hoped that this review will be appropriate for those undertaking these experiments for the first time or wishing to improve (or validate) a technique in what is frequently a confusing and contradictory field.

https://www.future-science.com/doi/pdf/10.2144/99261rv01

Quantitative RT-PCR: pitfalls and potential.

HNF-4 (hepatocyte nuclear factor 4) is a protein enriched in liver extracts that binds to sites required for the transcription of the genes for transthyretin (TTR), the carrier protein in the serum for vitamin A and thyroid hormone, and for apolipoprotein CIII (apoCIII), a major constituent of chylomicrons and very low-density lipoproteins (VLDL) Synthetic oligonucleotides derived from amino acid sequence of affinity-purified HNF-4 protein (54 kD) were used in the polymerase chain reaction (PCR) to isolate a cDNA clone encoding the protein HNF-4 is a member of the steroid hormone receptor superfamily with an unusual amino acid in the conserved "knuckle" of the first zinc finger (DGCKG) Studies with in vitro-translated HNF-4 protein show that it binds to its recognition site as a dimer, and cotransfection assays indicate that it activates transcription in a sequence-specific fashion in nonhepatic (HeLa) cells Northern blot analysis reveals that HNF-4 mRNA is present in kidney and intestine, as well as liver, but is absent in other tissues DNA-binding and antisera reactivity data suggest that HNF-4 could be identical to liver factor A1 (LF-A1), a DNA-binding activity implicated in the regulation of transcription of the alpha 1-antitrypsin, apolipoprotein A1, and pyruvate kinase genes The similarity between HNF-4 and other ligand-dependent transcription factors raises the possibility that HNF-4 and the genes it regulates respond to an as yet unidentified ligand

http://genesdev.cshlp.org/content/4/12b/2353.full.pdf

Liver-enriched transcription factor HNF-4 is a novel member of the steroid hormone receptor superfamily.

Transgenetic studies implicate interactions between homologous PrP isoforms in scrapie prion replication.

Background When the myocardium is deprived of blood, a process of ischemia, infarction, and myocardial remodeling is initiated. Hypoxia-inducible factor 1 (HIF-1) is a transcriptional activator of vascular endothelial growth factor (VEGF) and is critical for initiating early cellular responses to hypoxia. We investigated the temporal and spatial patterns of expression of the α subunit of HIF-1 (HIF-1α) and VEGF in specimens of human heart tissue to elucidate the early molecular responses to myocardial hypoxia. Methods Ventricular-biopsy specimens from 37 patients undergoing coronary bypass surgery were collected. The specimens were examined by microscopy for evidence of ischemia, evolving infarction, or a normal histologic appearance. The specimens were also analyzed with the reverse-transcriptase polymerase chain reaction for HIF-1α and VEGF messenger RNA (mRNA) expression and by immunohistochemical analysis for the location of the HIF-1α and VEGF proteins. Results HIF-1α mRNA was detected in myocardial ...

Puissant C

Papers

An improvement of the single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction.