For quantitative reverse transcriptase polymerase chain reaction (RT-PCR), 600 ng of total RNA was reverse transcribed by using the SuperScript III First-Strand Synthesis SuperMix (Thermo Fisher Scientific, #11752-05) according to the manufacturer’s instructions. After reverse transcription, RT-PCR was performed on 30 ng complementary DNA using SensiMix II Probe (Bioline, BIO-83005) and TaqMan Primer Assays (Thermo Fisher Scientific) for HPRT-VIC, RIP1K, RIP3K, and mixed lineage kinase domain-like protein (MLKL) on a CFX96 Real Time Detection System (Bio-Rad). Normalized expression to HPRT was quantified by using the comparative 2ΔΔCt method.
Sensimix 2 probe
Manufactured by Meridian Bioscience
SensiMix II Probe is a ready-to-use, high-performance real-time PCR mix designed for probe-based detection and quantification. It contains all the necessary components for efficient and sensitive real-time PCR, including a hot-start DNA polymerase, dNTPs, MgCl2, and stabilizers.
Automatically generated - may contain errors
Lab products found in correlation
Superscript 3 first strand synthesis supermix, by Thermo Fisher Scientific (1 mentions)
Taqman primer assays, by Thermo Fisher Scientific (1 mentions)
Cfx96 real time detection system, by Bio-Rad (1 mentions)
Custom taqman assay design tool, by Thermo Fisher Scientific (1 mentions)
Random primer, by Thermo Fisher Scientific (1 mentions)
Realplex2, by Eppendorf (1 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
Sensimix sybr hi rox kit, by Meridian Bioscience (1 mentions)
Mirneasy micro kit, by Qiagen (1 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (1 mentions)
7900ht fast real time pcr system, by Thermo Fisher Scientific (1 mentions)
Taqman gene expression assay, by Thermo Fisher Scientific (1 mentions)
4 protocols using sensimix 2 probe
1
Quantitative RT-PCR for Cell Death Regulators
2
Genotyping L1014F Vgsc Mutations
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Two assays to genotype the L1014F-Vgsc mutations in exon 20 of the Vgsc gene (see below), which has been implicated in resistance to pyrethroids and organochlorine insecticides were initially designed and applied in parallel to detect two non-synonymous variants; one to genotype TTA/TTT alleles and the other to detect TTA/TTC variants. Primer sets and TaqMan probes were designed using the Custom TaqMan® Assay Design Tool (Life Technologies).
TaqMan allelic discrimination reactions were carried out using approximately 20 ng of gDNA, 1x SensiMix II probe (Bioline), 0.4 µM of each primer (Kdr-F: 5′-CTTGGCCACCGTAGTGATAGG-3′ and Kdr-R: 5′-GCTGTTGGCGATGTTTTGACA-3′) and 0.1 µM of each probe (Probe-TTC-allele: 5′- FAM-CACGACGAAATTT-3′ or Probe-TTT-allele: 5′-FAM-TCACGACAAAATTT-3′ and Probe-TTA-allele/wildtype: 5′-VIC-ACTCACGACTAAATTT-3′), in a final volume of 10 µl. The PCR was performed on a Stratagene MX3005P with cycling parameters of 95 °C for 10 min followed by 40 cycles of 95 °C for 10 sec and 60 °C for 45 sec.
TaqMan allelic discrimination reactions were carried out using approximately 20 ng of gDNA, 1x SensiMix II probe (Bioline), 0.4 µM of each primer (Kdr-F: 5′-CTTGGCCACCGTAGTGATAGG-3′ and Kdr-R: 5′-GCTGTTGGCGATGTTTTGACA-3′) and 0.1 µM of each probe (Probe-TTC-allele: 5′- FAM-CACGACGAAATTT-3′ or Probe-TTT-allele: 5′-FAM-TCACGACAAAATTT-3′ and Probe-TTA-allele/wildtype: 5′-VIC-ACTCACGACTAAATTT-3′), in a final volume of 10 µl. The PCR was performed on a Stratagene MX3005P with cycling parameters of 95 °C for 10 min followed by 40 cycles of 95 °C for 10 sec and 60 °C for 45 sec.
3
Quantitative Analysis of RasGRP Expression
4
Analyzing Microglial Activation via qRT-PCR
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Striata were dissected in RNAse-free conditions. Total RNA was extracted according to the standard miRNeasy Micro kit protocol (Qiagen). Next, 350 ng of total RNA was reverse-transcribed using High-Capacity cDNA Reverse Transcription Kit (Applied Biosystem), and 10 ng of complementary DNA (cDNA) was amplified with SensiMix SYBR Hi-Rox Kit (Bioline; Meridian Life Science) in triplicate using the Applied Biosystem 7900HT Fast Real-Time PCR system. Messenger RNA (mRNA) relative quantification was performed using the comparative cycle threshold (2−ΔΔCt) method. β-actin was used as endogenous control.
Primer sequences:
Ionized calcium binding adaptor protein-1 (Iba-1, NM_019467): 5′-GACAGACTGCCAGCCTAAGACAA-3′ (sense), 5′-CATTCGCTTCAAGGACATAATATCG-3′ (antisense);
β-ACTIN (NM_007393): 5′-CCTAGCACCATGAAGATCAAGATCA-3′ (sense), 5′-AAGCCATGCCAATGTTGTCTCT-3′ (antisense).
For CD3+ mRNA detection, 17.5 ng of the same cDNA was amplified with SensiMix II Probe (Bioline; Meridian Life Science) by using TaqMan gene expression assays (ID Mm00599684_g1 and ID Mm00607939; Applied Biosystem).
Primer sequences:
Ionized calcium binding adaptor protein-1 (Iba-1, NM_019467): 5′-GACAGACTGCCAGCCTAAGACAA-3′ (sense), 5′-CATTCGCTTCAAGGACATAATATCG-3′ (antisense);
β-ACTIN (NM_007393): 5′-CCTAGCACCATGAAGATCAAGATCA-3′ (sense), 5′-AAGCCATGCCAATGTTGTCTCT-3′ (antisense).
For CD3+ mRNA detection, 17.5 ng of the same cDNA was amplified with SensiMix II Probe (Bioline; Meridian Life Science) by using TaqMan gene expression assays (ID Mm00599684_g1 and ID Mm00607939; Applied Biosystem).
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