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Use of Cytokines in Dermatotoxicology
Published in Francis N. Marzulli, Howard I. Maibach, Dermatotoxicology Methods: The Laboratory Worker’s Vade Mecum, 2019
G. Frank Gerberick, Elizabeth E. Sikorski, Cindy A. Ryan, Laura C. Limardi
Total RNA is isolated by acid guanidinium-thiocyanate-phenol-chloroform extraction (Chomczynski and Sacchi, 1987) or by using the RNeasy method from Qiagen (Chatsworth, California). The final preparation for both methods should be free of DNA and proteins and have a 260/280 ratio of approximately 1.7 to 2.0. RNA integrity is determined by denaturing gel electrophoresis.
Experimental models and measurements to study cardiovascular physiology
Published in Neil Herring, David J. Paterson, Levick's Introduction to Cardiovascular Physiology, 2018
Neil Herring, David J. Paterson
In terms of mRNA, the presence and level of expression can be evaluated using reverse transcription followed by polymerase chain reaction (PCR) with detection of the resulting complementary DNA (cDNA) products. The technique was developed by Mullis and Smith who received the Nobel Prize in Chemistry for their work in 1993. The first step requires the isolation of RNA from tissue (e.g. by homogenization and acid-guanidinium thiocyanate-phenol-chloroform extraction) and quantification of the amount of RNA extracted. A set amount of RNA is then reverse transcribed to cDNA. The cDNA is used as a template for exponential amplification with PCR guided by primers complementary to the three prime ends of the sense and antisense strands of the cDNA of interest. As well as primers, PCR requires a DNA polymerase with a temperature optimum at around 70°C, deoxynucleoside triphosphate nucleotides for cDNA synthesis and buffer solution with appropriate concentration of monovalent and bivalent cations. PCR then undergoes 20-40 amplification cycles, each with several temperature steps in a thermal cycling machine. First, there is a high temperature denaturation step (e.g. 94 °C-98 °C), followed by an annealing temperature for the primers to bind (e.g. 50 °C-65 °C), and then an extension stage at which DNA polymerase synthesizes the DNA product (around 70°C) as shown in Figure 19.7. This enables very small amounts of a specific mRNA to be detected. (Northern blotting requires alarge amount of mRNA to start with.) Using real-time PCR with fluorescent DNA labelling techniques, the amount of cDNA over each amplification cycle can be measured, allowing a degree of indirect quantification of the differences in starting mRNA to be assessed as shown in Figure 19.8. The exponential amplification during the multiple cycles of PCR can produce inaccurate end-point quantification because of the difficulty in maintaining linearity through the cycles. Samples can also become contaminated or degraded due to the presence of RNases.
Cardioprotective Efficacy of Coriandrum sativum (L.) Seed Extract in Heart Failure Rats Through Modulation of Endothelin Receptors and Antioxidant Potential
Published in Journal of Dietary Supplements, 2020
Neha Dhyani, Adila Parveen, Aisha Siddiqi, M. Ejaz Hussain, Mohammad Fahim
The expression of ETA and ETB receptors mRNA was analyzed by RT-PCR. The expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA was determined as an internal control. Total tissue RNA was isolated by acid guanidinium thiocyanate-phenol-chloroform extraction with Isogen according to the manufacturer’s instructions. The tissue was homogenized in Trizol (0.2 g tissue per 2 ml Trizol) with a tissue homogenizer. Chloroform extraction, isopropanol precipitation, and 75% (v/v) ethanol washing of precipitated RNA were subsequently performed. The obtained RNA was resolved in diethyl pyrocarbonate–treated water. The RNA concentration was measured using nanodrop. Total RNA (5 µg) was primed with 0.05 µg oligo-d(T) 12–18 and reverse transcribed by avian myeloblastosis virus reverse transcriptase using the First-Strand cDNA Synthesis Kit. The obtained cDNA was diluted in a 1:10 ratio, and 1 µl was used for real-time PCR.
The protective effect of Satureja bachtiarica hydroalcoholic extract on streptozotocin‐induced diabetes through modulating glucose transporter 2 and 4 expression and inhibiting oxidative stress
Published in Pharmaceutical Biology, 2019
Reyhaneh Joudaki, Mahbubeh Setorki
Samples of rat liver were frozen in an adequate volume of acid guanidinium thiocyanate solution and kept at −80 °C until RNA extraction. Total cellular RNA was extracted by the method of acid guanidinium thiocyanate phenol/chloroform extraction. The total tissue RNA concentration was measured by spectrophotometric absorbance (260 nm), and the quality of isolated RNA was verified by agarose gel electrophoresis with ethidium bromide staining. Purified total RNA (1 µg) was used as a substrate for reverse transcription. The reaction was performed by incubation of RNA with 1 µM oligo (dT) and 200 units of MMLV reverse transcriptase from a Clontech (Mountain View, CA) first strand cDNA synthesis kit. An aliquot (5 µL of a 1/10 dilution) of the cDNA of each sample was used for RT-PCR. The PCR primers used are shown in Table 1. DNA amplification was carried out in 1 × Taq polymerase buffer, 1.5 mM MgCI2 supplemented with 50 µM dNTP, 0.25 µM of 5′ and 3′-specific primers, 1 µCi of [α-32p] and 2 units of Taq polymerase (Promega C) in a final volume of 50 µL. The mixture was overlaid with mineral oil and amplified for 30 cycles (each consisting of denaturation for 1 min at 94 °C, annealing for 1 min at 60 °C, extension for 1 min at 72 °C) then extension for 7 min at 72 °C and storage at 4 °C in a Triothermoblock. cDNA products (10 µL) were size-separated by electrophoresis on a 10% acryl/bisacrylamide gel and stained with ethidium bromide (15 µg/mL). Each band was excised from the gel, and the quantity of 32p incorporated was measured in a scintillation counter (Weinstein et al. 1994).