Total RNA was extracted using the RNeasy minikit (Qiagen, GmBH, Hilden, Germany) and underwent column DNase I digestion (Qiagen) as recommended by the manufacturer. cDNA was synthesized from RNA in accordance with the manufacturer's instructions (qScript cDNA supermix; Quanta Biosciences, Gaithersburg, MD). Twenty nanograms of RNA equivalent DNA was used in triplicate. Quantitative reverse transcription-PCR (qRT-PCR) was performed using SsoFast EvaGreen supermix (Bio-Rad Laboratories, Hercules, CA), template DNA, and 500 nM each primer RT-KPC-F/RT-KPC-R, K. oxytoca rpoB-F/rpob-R, or K. pneumoniae rpoB-F/rpoB-R. Relative quantification of gene expression was determined using the averaged cycle threshold (CT) values for each isolate using the Pfaffl method (33 (link)). This equation uses an expression ratio to normalize the expression levels of blaKPC to the transcriptional level of the constitutively expressed rpoB gene.
Column dnase 1 digestion
Manufactured by Qiagen
Sourced in Germany, United States
Column DNase I digestion is a laboratory equipment product that serves the core function of removing DNA from RNA samples during RNA purification processes. It is designed to provide a controlled and effective method for the removal of contaminating DNA, ensuring the purity of the RNA samples for downstream applications.
Automatically generated - may contain errors
Lab products found in correlation
Rneasy mini kit, by Qiagen (1 mentions)
Ssofast evagreen supermix, by Bio-Rad (1 mentions)
Qscript cdna supermix, by Quanta Biosciences (1 mentions)
Taqman reagent, by Thermo Fisher Scientific (1 mentions)
Qiaamp dna kit, by Qiagen (1 mentions)
Rneasy 96 qiacube ht kit, by Qiagen (1 mentions)
Qx200 ddpcr system, by Bio-Rad (1 mentions)
2 protocols using column dnase 1 digestion
1
Quantification of KPC Gene Expression
2
Quantifying rAAV Genome Copy Number
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Mouse cornea genomic DNA was isolated using the QIAamp DNA kit (QIAGEN, Hilden, Germany) following manufacturer’s instructions and then digested with >10 U/μg SalI (New England Biolabs, Ipswich, MA, USA) at 37°C for 1 hr. There are two SalI sites in the rAAV genome, and SalI digestion ensures single copy emulsion for droplet digital PCR (ddPCR) quantification. Multiplexed ddPCR was performed on a QX200 ddPCR system (Bio-Rad Laboratories, Hercules, CA, USA) using TaqMan reagents targeting EGFP (catalog no. 4400293; Life Technologies) and the reference gene, transferrin receptor (Tfrc) (catalog no. 4458367; Invitrogen, Waltham, MA, USA). rAAV genome copy numbers per diploid genome were calculated as EGFP transgene copy numbers per two Tfrc gene copies.
Total RNA was extracted using the RNeasy 96 QIAcube HT kit with on-column DNase I digestion (QIAGEN, Valencia, CA, USA), reverse-transcribed into cDNA, and subjected to multiplexed ddPCR using TaqMan reagents targeting EGFP and Glyceraldehyde-3-Phosphate Dehydrogenase (gapdh) (catalog no. 4352339E; Life Technologies). The quantity of EGFP was normalized to gapdh levels.
Total RNA was extracted using the RNeasy 96 QIAcube HT kit with on-column DNase I digestion (QIAGEN, Valencia, CA, USA), reverse-transcribed into cDNA, and subjected to multiplexed ddPCR using TaqMan reagents targeting EGFP and Glyceraldehyde-3-Phosphate Dehydrogenase (gapdh) (catalog no. 4352339E; Life Technologies). The quantity of EGFP was normalized to gapdh levels.
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