RNA extraction was performed using the Qiagen RNeasy Plus minispin column kit, eluting in 50 µL of RNase-free water, and using an additional DNase step. RNA concentration was determined using the Qubit RNA High-Sensitivity kit (Life Technologies). Reverse transcription was performed using the Invitrogen SuperScript III kit according to the manufacturer's instructions. TaqMan qPCR and TaqMan LDA card assays were performed using TaqMan Universal PCR Master Mix and assays (Applied Biosystems) according to the manufacturer's guidelines. Results were normalized to the housekeeping gene Gapdh and analyzed with HTqPCR (Dvinge and Bertone 2009 (link)). The following TaqMan assays were used: mGapdh (Mm99999915_g1), mFoxG1 (Mm02059886_s1), mFoxo3 (Mm01185722_m1), mGfap (Mm01253033_m1), mAqp4 (Mm00802131_m1), mS100b (Mm00485897_m1), mNestin (Mm00450205_m1), mOlig2 (Mm01210556_m1), mBlbp (Fabp7) (Mm00445225_m1), mSox2 (Mm03053810_s1), mDnmt1 (Mm01151063_m1), mDnmt3b (Mm01240113_m1), mTet3 (Mm00805756_m1), and hGAPDH (Hs02758991_g1).
Taqman universal pcr master mix and assays
Manufactured by Thermo Fisher Scientific
Sourced in United States
TaqMan Universal PCR Master Mix and assays are designed for use in real-time quantitative PCR (qPCR) experiments. The master mix provides the necessary reagents, including DNA polymerase, dNTPs, and buffer, to enable efficient and reliable DNA amplification. The assays include pre-designed and validated primer and probe sets for specific gene targets, allowing for accurate and sensitive detection and quantification of target sequences.
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2 protocols using taqman universal pcr master mix and assays
1
Comprehensive Gene Expression Analysis in Mouse Tissues
2
Quantification of Inflammasome Transcripts
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The NLRP1, NLRP3 and AIM2 quantification of the transcription was carried out on the 7500 real-time PCR system using the Taqman® Universal PCR Master Mix and Assays on demand (Applied Biosystems, Carlsbad, USA). Extracted RNA from isolated tissues was initially reverse-transcribed using a High-Capacity cDNA Archive Kit (Applied Biosystems). Primers used to determine inflammasome mRNA levels (forward primer, reverse primer) were 5′-CAACAA GACTTGAACACAACGAG-3′ and 5′-CTCT CAATGACTGTGCTGGGTA-3′ for NLRP1; 5′-GATCTTCGCTGCGATCAACAG-3′ and 5′-CGTGCATTATCTGAACCCCAC-3′ for NLRP3; and 5′-AGCTGACATCTGGAGTTC ATAGC-3′ and 5′-CTGCTTAGACCAGTTG GCTTG-3′ for AIM2.
Relative quantification of the NLRP3, NLRP1 and AIM2 expression was carried out using the ΔΔCT (threshold cycle) comparative method. 14 The assay was run in triplicate for each case to avoid possible technical variability.
Relative quantification of the NLRP3, NLRP1 and AIM2 expression was carried out using the ΔΔCT (threshold cycle) comparative method. 14 The assay was run in triplicate for each case to avoid possible technical variability.
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