Mouse tissues, blood, or cultured cells were collected for total RNA extraction with TRIreagent (Molecular Research Center, Inc.) and converted into first strand cDNA using the iSCRIPT™ cDNA synthesis kit (Bio-Rad). qPCR assays were performed using iTAQ™ polymerase supermix for probe-based assays (Bio-Rad) or iQ™ SYBR® Green Supermix polymerase (Bio-Rad). WNV-envelope (WNV-E) gene and mouse gene primers and probes sequences were adapted according to previous publications: WNV-E (4 (link)), β-Actin (42 (link)), Tlr7 (24 (link)), Irf-7 (43 (link)), Ifn-α (43 (link)), Isg-56 (43 (link)), Isg-54 (44 (link)), Isg-49 (44 (link)), Ifn-β (43 (link)), and Socs-1 (45 (link)). Primers were designed for murine Bax, forward 5’-TGCTAGCAAACTGGTGCTCA-3’ and reverse 5’-TAGGAGAGGAGGCCTTCCCAG-3’. Data were presented either as relative fold change (RFC) by the 2−ΔΔCT method, using β-actin as a housekeeping gene, or was expressed as a ratio of target gene to β-actin copy numbers. All the primers and probes were synthesized either by Integrated DNA Technologies or Applied Biosystems.
Itaq polymerase supermix for probe based assays
Manufactured by Bio-Rad
ITAQ™ polymerase supermix is a ready-to-use solution for probe-based qPCR assays. It contains a highly-processive DNA polymerase, dNTPs, MgCl2, and stabilizers optimized for sensitive and specific detection of target sequences.
Automatically generated - may contain errors
2 protocols using itaq polymerase supermix for probe based assays
1
Quantitative PCR Analysis of Virus-Induced Host Response
2
Quantitative PCR for Viral RNA Detection
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total RNA was extracted from tissues or cultured cells by using TRIreagent (Molecular Research Center, Inc.) and converted to cDNA using iSCRIPT cDNA synthesis kit (Bio-Rad). QPCR assays were performed using iTAQ polymerase supermix for probe-based assays (Bio-Rad) or iQ SYBR Green Supermix (Bio-Rad). Viral RNA of ZIKV envelope (E) (Acharya et al., 2016 (link)) was measured by qPCR and infectious viruses were measured by plaque assay as we previously described (Acharya et al., 2015 (link)). Threshold cycle values that were ≥39 cycles were excluded from the qPCR results, and 1 PFU per volume of sample was set as the limit of viral detection for the plaque assays. WNV-envelope (E) gene primers and probes sequences were adapted according to a previous publication (Town et al., 2009 (link)). All additional gene primer sequences are described in Supplementary Table S1 .
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!