PCR primers obtained from the qPrimerDepot database55 (link) or designed with Primer Designing Tool (https://www.ncbi.nlm.nih.gov/tools/primer-blast/ ) were chosen overlapping an intron or hybridizing an exon junction when possible and checked for in silico specificity before synthesis by Eurofins Operon (Supplementary Table S1 ). qPCR analysis was done in triplicate 15-µL reactions with SYBR Premix Ex Taq Tli RNase H Plus mix (Takara) including 5 µL of 30-fold diluted cDNA and 0.5 µM of each primer. A 2-steps PCR program with hybridization at 61 °C followed by specificity check on fusion curves, was performed on CFX384 thermal cycler (Bio-Rad Laboratories). Quantitative values were normalized using the NORMA-Gene method56 (link) for whole-skin data and with GAPDH and ACTB housekeeping genes for microdissection data57 (link). Relative expression ratios were calculated by the delta-delta Cq method58 (link), based on a theoretical efficiency considered at 100%, using as calibrator the untreated condition of similar duration as treated samples (day 10).
Sybr premix ex taq tli rnase h plus mix
Manufactured by Takara Bio
Sourced in Germany
SYBR Premix Ex Taq (Tli RNase H Plus) is a ready-to-use master mix for real-time PCR. It contains SYBR Green I dye, Taq DNA polymerase, and buffer components necessary for real-time PCR amplification and detection.
Automatically generated - may contain errors
Lab products found in correlation
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (3 mentions)
Rnaiso plus reagent, by Takara Bio (3 mentions)
Lightcycler 480, by Roche (2 mentions)
Cfx384 thermal cycler, by Bio-Rad (1 mentions)
Lightcycler 480 instrument, by Roche (1 mentions)
Lightcycler 480, by Meridian Bioscience (1 mentions)
Sensifast probe lo rox mix, by Meridian Bioscience (1 mentions)
4 protocols using sybr premix ex taq tli rnase h plus mix
1
Quantitative PCR Analysis of Gene Expression
2
qPCR Analysis of Gene Expression
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The RNA was extracted from the cells using RNAiso Plus reagent (TaKaRa, Shimogyō-ku, Kyoto, Japan), and reverse transcription (RT) was performed using random primers with the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Waltham, MA, USA). The qPCR reaction was performed using the SYBR Premix Ex Taq (Tli RNase H Plus) mix (TaKaRa) in a LightCycler 480 instrument (Roche, Penzberg, Upper Bavaria, Germany). The fold change in gene expression was calculated using the comparative crossing point method (2-∆∆CP). Primers and probes used for qPCR are listed in Table 1 ; gene expression was normalized to that of glyceraldehyde-3-phosphate dehydrogenase (GAPDH).
3
Quantitative RT-PCR Analysis of Gene Expression
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RNA was extracted from cells using RNAiso Plus reagent (TaKaRa) and reverse transcribed with random primers using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems). Real-time PCR reactions were performed using SYBR Premix Ex Taq (Tli RNase H Plus) mix (Takara) in a LightCycler 480 instrument (Roche). Primers used in this study are listed in Supplementary Table 2 and gene expression was normalized to that of glyceraldehyde-3-phosphate dehydrogenase (GAPDH).
4
Quantitative Analysis of EBV Transcripts and Viral Load
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RNA was extracted from cells using RNAiso Plus reagent (TaKaRa) and reverse transcribed with gene-specific reverse transcription (RT) primers or random primers using a high-capacity cDNA reverse transcription kit (Applied Biosystems). The real-time PCR was performed using SYBR Premix Ex Taq (Tli RNase H Plus) mix (TaKaRa) in a LightCycler 480 instrument (Roche). Primers used to amplify the alternatively spliced BART RNAs, BARF0, RPMS1, RPMS1A, and A73, are listed in Table 1 , and gene expression was normalized to that of glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The real-time PCR amplification was performed with miRNA-specific TaqMan probes using SensiFAST Probe Lo-ROX mix (Bioline) in a LightCycler 480 instrument. Primers and probes used for BART miRNA detection were described previously (37 (link)). To determine EBV copy number, a 75-bp fragment of the BamH-W region (GenBank accession number V01555.2 ; nucleotides 17721 to 17796) was amplified by quantitative PCR (qPCR) from phenol-chloroform-extracted DNA. The same fragment was cloned into the TA cloning vector pMD18-T, which was then serially diluted to obtain a standard curve.
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