TRIzol (1 ml) was added to the wells and total RNA was extracted from the cells according to the manufacturer’s protocol (Invitrogen; Thermo Fisher Scientific, Inc.). The total RNA (1 µg) was reverse transcribed to cDNA using a SuperScript™ II Reverse Transcriptase kit (Invitrogen; Thermo Fisher Scientific, Inc.). The temperature protocol was as follows: 42°C for 2 min, and 4°C for 30 min; followed by 37°C for 15 min at 85°C for 5 sec, and 4°C for 30 min. These generated cDNA samples were then amplified with RT-qPCR in 20 µl of the reaction system, which contained the 10 µl SYBR® Primix Ex Taq™ (Takara Bio, Inc., Otsu, Japan), 2 µl cDNA, 0.4 µl each primer and 7.2 µl RNase-free H2O, following the manufacturer’s protocol. RT-qPCR analysis was performed on a Roche Light Cycler® 480 (Hoffmann-La Roche Ltd., Basel, Switzerland). The thermal cycling conditions were as follows: 95°C for 30 sec, followed by 40 cycles of 95°C for 5 sec and 60°C for 20 sec. The relative gene expression was calculated using the 2−ΔΔCq method (33 (link)), normalizing with GADPH levels. The primers used were as follows: GAPDH, forward 5′-GCA CCG TCA AGG CTG AGA AC-3′ and reverse 5′-TGG TGA AGA CGC CAG TGG A-3′; TAZ, forward 5′-CCT CTT CAA TGA TGT AGA GTC TGC-3′ and reverse 5′-AGT GAT TAC AGC CAG GTT AGA AAG-3′.
Sybr primix ex taq
Manufactured by Takara Bio
Sourced in Japan
SYBR Primix Ex Taq is a ready-to-use PCR master mix containing SYBR Green I dye, Taq DNA polymerase, and necessary reagents for real-time PCR amplification.
Automatically generated - may contain errors
Lab products found in correlation
M mlv reverse transcriptase, by Promega (2 mentions)
Nd 1000 spectrophotometer, by Thermo Fisher Scientific (2 mentions)
Superscript 2 reverse transcriptase kit, by Thermo Fisher Scientific (1 mentions)
Lightcycler 480, by Roche (1 mentions)
Total rna purification kit, by Tiangen Biotech (1 mentions)
Steponeplus real time pcr system, by Thermo Fisher Scientific (1 mentions)
Miniopticon system, by Bio-Rad (1 mentions)
Trizol reagent, by Takara Bio (1 mentions)
Random oligonucleotide primers, by Promega (1 mentions)
Topscripttm rt drymix, by Enzynomics (1 mentions)
Easy blue kit, by iNtRON Biotechnology (1 mentions)
Cdna reverse transcription kit, by Thermo Fisher Scientific (1 mentions)
Cfx96tm real time pcr detection system, by Bio-Rad (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Icycler, by Takara Bio (1 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
Abi prism 7500, by Thermo Fisher Scientific (1 mentions)
Primescript rt reagent kit, by Takara Bio (1 mentions)
8 protocols using sybr primix ex taq
1
Total RNA Extraction and RT-qPCR Analysis
2
Quantitative Analysis of Corneal Gene Expression
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Corneal tissues (n = 4 samples per group) and cells total RNA was extracted using a total RNA purification kit (TianGen, Beijing, China). The amount of total RNA was quantified by spectrophotometry and cDNA was synthesized with M-MLV reverse transcriptase (Promega, Madison, USA). Real-time quantitative PCR (RT‒qPCR) was performed with SYBR Primix Ex Taq (Takara, Japan) and the StepOnePlus Real-Time PCR system (Applied Biosystems, CA, USA) according to the manufacture’s protocol. The relative quantity of mRNA or miR-146a with regard to the expression of GAPDH or U6 was estimated using the 2−△△Ct method.
3
Epididymal Gene Expression Analysis
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Total RNA was extracted from the caput, corpus, and cauda regions of the rat epididymis, using TRIzol Reagent (Takara, Shiga, Japan) according to the supplier instructions, and semi-quantitative RT-PCR for RANTES, CCR1, CCR3 and CCR5 were performed as described previously (36 (link)). Real-time quantitative PCR analysis of V-ATPase, RANTES, CCR1 and CCR5, iNOS, AGTR2 expression in the different epididymal regions were performed with the MiniOpticon system (Bio-Rad, Hercules, CA, United States). Each reaction was performed in triplicate by using 10 ng of cDNA from each sample and SYBR Primix Ex Taq (TaKaRa, Shiga, Japan). The relative abundance of target transcript was quantified using the comparative Ct method. The β -actin or GAPDH from the same exacts were used as internal control. Data obtained from three independent experiments was calculated. Primers for PCR and qPCR were designed according to the rat sequences found in GenBank (Supplementary Tables 1 , 2 ).
4
Quantitative Real-Time PCR Analysis of Gene Expression
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Total cellular RNA was extracted by using Easy Blue® kits (Intron Biotechnology, Seoul, Korea). RNA (1 μg) was reverse-transcribed (RT) using 0.5 mg/mL random oligonucleotide primers (Promega, Madison, WI, USA) and TOPscriptTM RT DryMIX (Enzynomics, Daejeon, Korea). PCR amplification was performed using the incorporation of SYBR green using SYBR Primix Ex Taq (TaKaRa Bio Inc., Shiga, Japan). The PCR primers used in this study are described in Table S1 . Steady-state mRNA levels were determined by real time qPCR using the TaKaRa thermal cycler device. Mean Ct values of genes were calculated from triplicate measurements and normalized versus the mean Ct of GAPDH. The PCR primers used in this study are described in Table S1 .
5
Cardiac Gene Expression Profiling
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The alteration of gene expression levels in cardiac tissues, including peroxisome proliferator-activated receptor gamma co-activator 1a (PGC-1α) [2 (link)], mitochondrial transcription factor (mtTFA) [2 (link)], nuclear respiratory factor 1 (NRF1) [2 (link)], C/EBP homologous protein (CHOP) [19 (link)], activating transcription factor 4 (ATF4) [19 (link)], Bcl-2 [20 (link)], and Bax [20 (link)], was measured using a quantitative real-time polymerase chain reaction (qRT-PCR). Trizol reagent (Invitrogen, Cergy Pontoise, France) was used according to the manufacturer’s protocol for the extraction of total RNA from the frozen left ventricles of cardiac tissues. cDNA reverse transcription kits (Applied Bio-systems, CA, USA) were used to synthesize cDNA from isolated total RNA. qRT-PCR was performed using cDNA with the SYBR Primix Ex Taq (TaKaRa, Kyoto, Japan) on a CFX96TM real-time PCR detection system (Bio-Rad Laboratories, Hercules, CA, USA). The reaction volume was 20 mL and the final working concentrations of all primers were 200 nmol/L. The obtained qPCR data were analyzed through the ΔΔCT method using a housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) by using Bio-Rad CFX Manager Ver. 2.1 software (Bio-Rad Laboratories, Hercules, CA, USA). Product specificity was confirmed by melting or dissociation curve analysis. Primer sequences are presented in Table 1 .
6
Quantification of ACE2 and TMPRSS2 mRNA
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Total RNA was extracted from cells and the human corneal epithelium using a Qiagen RNeasy Mini Kit according to the manufacturer's instructions and was reverse transcribed into cDNA using M-MLV reverse transcriptase (Promega). Real-time PCR was performed using a Bio-Rad iCycler with SYBR Primix Ex Taq (Takara) as described previously.19 (link) The primer sequences were as follows: ACE2 (forward, 5′-CGAGTGGCTAATTTGAAACCAAGAA-3′, reverse, 5′-ATTGATACGGCTCCGGGACA-3′)20 (link); TMPRSS2 (forward, 5′-ACTCTGGAAGTTCATGGGCAG-3′, reverse, 5′-TGAAGTTTGGTCCGTAGAGGC-3′)21 (link); GAPDH (Glyceraldehyde-3-Phosphate Dehydrogenase) (forward, 5′-TGCCCTCAACGACCACTTTG-3′, reverse, 5′- CTGGTGGTCCAGGGGTCTTA-3′). Gene expression was normalized to the GAPDH mRNA level.
7
Quantitative RT-PCR for Gene Expression
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Total RNA was extracted from AMC-HN-8 SH1, SH2, SH3, SH4, and NC cells using the RNA Extraction Reagent Kit (CWBiotech, China) in accordance with the manufacturer's instructions. Total RNA was reverse transcribed into cDNA using the PrimeScript RT Reagent Kit (Takara, Japan). Subsequently, qPCR was performed using SYBR Primix Ex Taq (Takara, Japan) and an RT-qPCR system (ABI PRISM 7500, Applied Biosystems, CA), which was also used to analyze the data. Recombinant ribosomal protein L13A (RPL13A) was chosen as the internal control. The 2-ΔΔCt method was used to calculate relative mRNA expression (DIAPH1/RPL13A). The same method was applied to FD-LSC-1 SH1, SH2, and NC cells using glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as the internal control. All primers used for RT-qPCR were designed and synthesized by Hanyin Co (Shanghai, China). The primers used for RT-qPCR were shown in Table 2 .
8
Quantitative Real-Time PCR Assay
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Total RNA was isolated with the RNAisol PLUS reagent (Takara Bio Inc.), according to the manufacturer's protocol. The concentration of total RNA was calculated from its absorbance at 260 nm and 280 nm, each with an ND1000 spectrophotometer (Thermo, USA). First-strand cDNA was synthesized with 1 μg of total RNA according to the manufacturer's protocol (Takara Bio Inc.). SYBR-Green-based quantitative real-time PCR was performed using SYBR Primix Ex Taq (Takara Bio Inc.) with the appropriate sense and antisense primers. The primer sets used in this study are shown in Table 1 . All reactions were carried out in triplicate and data were analyzed by the 2–ΔΔCT method. Beta-actin was used as an internal standard gene.
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