Total RNA was extracted from the brain tissue of C57BL/6J mice using ExtractRNA (Evrogen, Moscow, Russia), treated with RNA-free DNase (Promega, Madison, WI, USA), and diluted to 0.125 µg/µL with diethyl pyrocarbonate-treated water. One microgram of total RNA was subjected to cDNA synthesis with a random hexanucleotide mixture [42 (link),43 (link),44 (link)]. The number of cDNA copies for all studied genes was evaluated by qPCR on a LightCycler 480 (Roche Applied Science, Rotkreuz, Switzerland) with specific primers (Table 1 ), SYBR Green I fluorescence detection (R-414 Master mix, Syntol, Moscow, Russia), and 50, 100, 200, 400, 800, 1600, 3200, or 6400 copies of genomic DNA as external standards. The calibration curve in the coordinates Ct (threshold cycle value) and minus log P (decimal logarithm of the amount of DNA standard) was plotted automatically using the LightCycler 480 System software. Gene expression is presented as the relative number of cDNA copies per 100 copies of DNA-dependent RNA polymerase 2 subunit A (Polr2a) cDNA, which served as an internal standard [42 (link),43 (link),44 (link)]. A melting-curve analysis was performed at the end of each run for each primer pair, allowing us to control the amplification specificity.
Rna free dnase
Manufactured by Promega
Sourced in United States
RNA-free DNase is a recombinant enzyme that selectively degrades DNA without affecting RNA. It is designed for use in RNA-sensitive applications where complete removal of DNA is required.
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Lab products found in correlation
4 protocols using rna free dnase
1
Quantifying Gene Expression in Mouse Brain
2
Isolation and Analysis of Granulocyte and Monocyte RNA
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Total RNA of human granulocytes and monocytes was isolated using the RNeasy Mini Kit System (Qiagen, Venlo, The Netherlands), treated with RNA-free DNase (Promega, Madison, WI) and reverse transcribed using oligo(dT) primers and Moloney murine leukemia virus RT (Promega). Primers and RT-PCR conditions can be found in the online supplement (see Table S1, Supplemental Digital Content 1, at http://links.lww.com/SHK/A387 ). Data were analyzed using the comparative Ct method.
3
Quantitative PCR Analysis of Gene Expression
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Total RNA was extracted using the ExtractRNA reagent (Evrogen, Moscow, Russia) according to the manufacturer’s instructions, treated with RNA-free DNase (Promega, Madison, WI, USA), and diluted to 0.125 μg/μL with diethyl pyrocarbonate–treated water. One microgram of total RNA was subjected to cDNA synthesis with a random hexanucleotide mixture [104 (link),105 (link)]. The numbers of cDNA copies for gene encoding DNA-dependent RNA polymerase 2 subunit A (Polr2a), BDNF, receptors TrkB and p75, TH, DAT, MAOA, COMT, and DA receptors D1 and D2 were determined with quantitative PCR on LightCycler 480 (Roche Applied Science) using selective primers (Table A1 ), SYBR Green I fluorescence detection (R-414 Master mix, Syntol, Moscow, Russia), and genomic DNA extracted from the livers of male C57BL/6 mice as the external standard. We utilized 50, 100, 200, 400, 800, 1600, 3200, and 6400 copies of genomic DNA/μL as external standards for all genes under study. Reagent controls were set up under the same conditions but without the template. Gene expression was evaluated as the number of cDNA copies per 100 copies of Polr2a cDNA [104 (link),105 (link),106 (link)]. Melting-curve analysis was performed at the end of each run for each primer pair, allowing us to control the amplification specificity.
4
Quantifying PTPN5 Isoforms in Rat Retina
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Total RNA samples were extracted from 60 µL of the retinal homogenate using 300 µL of TRIzol Reagent (Ambion, Waltham, MA, USA) according to the manufacturer’s instructions. The extracted RNA samples were treated with RNA-free DNase (Promega, Madison, WI, USA) and diluted to 0.125 µg/µL with diethyl pyrocarbonate–treated water. The obtained total RNA was subjected to cDNA synthesis with a random hexanucleotide mixture (BioLab Mix, Novosibirsk, Russia). To evaluate the number of Ptpn5 cDNA copies, we performed SYBR Green I fluorescence detection (R-402 Master mix, Syntol, Novosibirsk,, Russia) with primers specific to STEP46 and STEP61 isoforms (forward primer: 5′-CGTGGTAGACATCCTAAAGACC-3′, reverse primer: 5′-CTGATACTGTTCGCATGTTTGG-3′) with respect to 100 copies of a housekeeping gene: DNA-dependent RNA polymerase II (Polr2a) (forward primer: 5′-TTGTCGGGCAGCAGAACGTG-3′, reverse primer: 5′-CAATGAGACCTTCTCGTCCTCCC-3′). As external standards, we used genomic DNA (0.5, 1, 2, 4, 8, 16, 32, and 64 ng/µL) extracted from the liver of male Wistar rats [51 (link),52 (link)]. To monitor amplification specificity, we performed melting curve analysis at the end of each run for each primer pair.
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