mRNA from T cells was extracted using Trizol as previously described [30] (link). First strand cDNA was synthesized by RSIII, according to the manufacturer's protocol (Invitrogen). Then, the first strand cDNA was used for quantitative real-time PCR using IQ SYBR Green (Invitrogen) on the MyiQ real-time detection system (Bio-Rad) following the manufacturer's protocol. The forward and reverse primers for Bcl-XL, Bcl-2, Bim, β-Actin and TCR Vβ13 have been previously described [39] (link), [40] (link). Experimental data was normalized to the control group using CFX manager software (Bio-Rad) following the manufacturer's protocol.
Myiq real time detection system
Manufactured by Bio-Rad
The MyiQ real-time detection system is a laboratory instrument designed for real-time PCR analysis. It functions by detecting and quantifying target DNA sequences in a sample during the amplification process. The system provides real-time monitoring of the reaction, allowing for precise quantification of the initial target amount.
Automatically generated - may contain errors
Lab products found in correlation
Iq sybr green, by Thermo Fisher Scientific (2 mentions)
Cfx manager software, by Bio-Rad (1 mentions)
Facsaria, by BD (1 mentions)
Rnaeasy kit, by Qiagen (1 mentions)
Sybr green pcr master mix, by Bio-Rad (1 mentions)
Superscript 2 reverse, by Thermo Fisher Scientific (1 mentions)
Faststart universal sybr green master rox, by Roche (1 mentions)
Rneasy kit, by Qiagen (1 mentions)
Qiaamp dna kit, by Qiagen (1 mentions)
Magnetic activated cell sorting (macs), by Miltenyi Biotec (1 mentions)
Iq sybr green supermix, by Bio-Rad (1 mentions)
Icycler, by Bio-Rad (1 mentions)
6 protocols using myiq real time detection system
1
Quantitative mRNA Analysis of T Cell Genes
2
Enriching and Profiling Type II NKT Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
T cells were enriched from Jα18-/- mouse liver or spleen lymphocytes using MACS negative selection as previously described. Type II NKT cells were sorted from pooled enriched T cells using a BD FacsAria. RNA was extracted from sorted cells using RNA-easy kit (QIAGEN) and cDNA was generated using Superscript II reverse transcriptase (Invitrogen). RT-PCR was performed using SYBR Green PCR master mix with cytokine primers (listed in Supplementary Table 1
3
Renal Inflammation Markers After IR
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Renal inflammation after IR was also assessed by measuring proinflammatory mRNA markers, including IL-6, ICAM-1, MCP-1, KC, MIP-2, and TNF-α qRT-PCR, as described previously with primers listed in Table 2 (46 (link)). Primer design was based on published GenBank sequences. To confirm equal RNA input, GAPDH mRNA and relative expression of proinflammatory mRNA were calculated with the ΔΔCt method. qRT-PCR was performed using MyiQ Real Time Detection System (Bio-Rad) using FastStart Universal SYBR Green Master (ROX) (Roche).
4
Quantifying Mitochondrial DNA in T Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Splenic CD4+ T cells from B6 mice and iNKT cells from Vα14Tg mice were positively selected using MACS (Miltenyi). Genomic DNA and mitochondrial DNA were extracted with QIAamp DNA kit (Qiagen) for determining mt DNA/nuclear DNA ratio. Total RNA from thymocytes, sorted DP thymocytes or splenocytes transfected with siRNAs were extracted using RNeasy kit (Qiagen). Following reverse transcription, PCR was performed using SYBR Green PCR master mix with respective primers (SI Appendix, Table S1 ) and MyiQ real-time detection system (Bio-Rad). Each PCR was run in duplicate or triplicate and normalized to β-actin.
5
Quantitative RT-PCR Analysis of Vaginal Gene Expression
6
qPCR Quantification of AAV-FVIII Viral Titers
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
qPCR was used to determine AAV-FVIII viral genome titers in anion exchange fractions from single-cell factories and in dialyzed CsCl fractions from triple-cell factories. Primers targeted the HLP promoter in the 5′ region of the viral genome, or the middle of the FVIII gene: 5′-CAGGACGCTGTGGTTTCTG-3′ (HLP forward), 5′-TGCCTGAAGCTGAGGAGAC-3′ (HLP reverse), 5′-GGAGATGAAGAAGGAGGACTTTG-3′ (FVIII forward), and 5′-TCCACAGCAGCAATGAAGTAG-3′ (FVIII reverse). Samples were run in duplicates of 25 μl in Bio-Rad iQ Sybr Green Supermix on the Bio-Rad iCycler with attached MyIQ real-time detection system (Bio-Rad Laboratories, Hercules, CA).`
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!