Total RNA was isolated from cultured cells using the RNAiso Plus reagent (TakaRa), and reverse transcription (RT) experiment was carried out using the HiScript II Q RT SuperMix for qRT-PCR kit (Vazyme). RT-PCR was carried out using the AceQ qPCR SYBR Green Master Mix (Vazyme) on a RT-PCR system (Roche) with primers as listed in Supplementary Table S3 . The expression levels of indicated genes were normalized to an internal control(18 S), and the relative expression levels were evaluated using the 2−ΔΔCT method. Each target was measured in triplicate.
Rt pcr system
Manufactured by Roche
Sourced in Switzerland
The RT-PCR system is a laboratory instrument used for the detection and quantification of specific genetic sequences through the process of reverse transcription-polymerase chain reaction (RT-PCR). It is a core tool for various applications in molecular biology, genomics, and diagnostic testing.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (2 mentions)
Trizol, by Thermo Fisher Scientific (2 mentions)
Hiscript 2 q rt supermix for qrt pcr kit, by Vazyme (1 mentions)
Rnaiso plus reagent, by Takara Bio (1 mentions)
Aceq qpcr sybr green master mix, by Vazyme (1 mentions)
Sybr green master mix, by Roche (1 mentions)
Quick rna miniprep kit, by Zymo Research (1 mentions)
Lc480 light cycler, by Roche (1 mentions)
Transscript one step gdna removal and cdna synthesis kit, by Transonic Systems (1 mentions)
Sybr premix ex taq tli rnaseh plus, by Takara Bio (1 mentions)
Transcriptor first strand cdna synthesis kit, by Roche (1 mentions)
Hrp conjugated secondary antibody sc 2004, by Santa Cruz Biotechnology (1 mentions)
Norrin, by R&D Systems (1 mentions)
Ab32572, by Abcam (1 mentions)
Sybr green pcr master mix, by Thermo Fisher Scientific (1 mentions)
Sybr green pcr master mix, by Vazyme (1 mentions)
Iq sybr green supermix, by Bio-Rad (1 mentions)
Trizol solution, by Thermo Fisher Scientific (1 mentions)
Primerscript rt reagent kit, by Takara Bio (1 mentions)
Sybr green pcr master mix, by EURx (1 mentions)
10 protocols using rt pcr system
1
Quantitative Real-Time PCR Analysis
2
Quantitative PCR of GABA Receptors
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qPCR was performed using a RT‐PCR system (Roche) with the SYBR Green Master mix (Roche). Each reaction was performed in a volume of 10 μL with 50 ng of cDNA template. All samples were analyzed three times and the reactions were performed for 40 cycles. Primers for the following genes were designed using perlPrimer software: Gabra1, Gabra6, Gabrg1, Gabrg2, Gabbr1, and Gapdh. Gene expression was standardized to the reference gene (in vivo/IVF) and calculated using the ΔΔCT method.
3
Quantifying a7nAChR Expression in Cells
4
Transcriptomic Analysis of Mouse Brain
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Total RNA from mice brain tissues or cultured cells was lysed using 1 mL of Trizol reagent according to the manufacturer's instructions (Invitrogen). RNA was then reverse transcribed into cDNA with TransScript One‐Step gDNA Removal and cDNA Synthesis Kit (TRANS). The cDNA was amplified using quantitative SYBR®Premix Ex Taq™ (Tli RNaseH Plus; Takara). Quantitative real‐time PCR (qPCR) was performed using the two‐step method RT‐PCR system (Roche). Each sample was performed in triplicate. mRNA levels were normalized to values for GAPDH using the 2−ΔΔCT method. qPCR primer sequences are listed in Appendix Table S2 (Appendix).
5
Quantifying Myostatin mRNA Expression
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After extraction and quantification of total RNA, myostatin (Mstn) mRNA expression levels were quantified using the Roche RT-PCR system. cDNA was synthesized using a Roche Transcriptor first strand cDNA synthesis kit following manufacturer’s guide. mRNA levels were quantified using LightCycler® 480 SYBR Green I master mix again following manufacturers guide. The relative expression of Mstn mRNA was determined using the LinRegPCR software program [24 (link)] and normalized to GAPDH expression. GAPDH (237 bp) and Mstn (167 bp) products were confirmed by agarose gel electrophoresis.
6
Wnt Pathway Signaling Regulation
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TriZol was purchased from Invitrogen (NY, US). Reverse Transcription and RT-PCR System were purchased from Roche (Mannheim, Germany) and Takara (Shiga, Japan), respectively. All other reagents were obtained from Sigma (St. Louis, MO). For western blot analyses and immunocytochemistry, a rabbit polyclonal antibody against the humanWnt2b receptor (ab50575), a polyclonal antibody against human Fzd5 (ab14475) and a monoclonal antibody against β-catenin (E247) (ab32572) were obtained from Abcam (MA, US), and an antibody to rat GAPDH was obtained from Epitomics (2251-1, CA, US). The HRP-conjugated secondary antibody (sc-2004) for western blotting was from Santa Cruz Biotechnology (CA, USA). The proteins DKK-1 (a Wnt-pathway inhibitor) and Norrin (a Wnt-pathway activator) were purchased form R&D Systems (Minneapolis, MN, USA).
7
Quantifying Gene Expression in Virus-Infected Cells
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RNA was isolated from RD and Vero cells using TRIzol reagent (15596026, Invitrogen™). The transcription of various genes was quantified using SYBR Green master mix. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) served as the internal control. qPCR was carried out using a reaction volume of 0.02 mL in a RT-PCR system (Roche, Switzerland) and SYBR Green PCR master mix (Thermo Fisher Scientific). qPCR was conducted at 95 °C for 10 min, followed by 40 cycles of 60 °C for 15 s and 72 °C for 30 s. Copy number of the target genes was evaluated using the comparative CT approach (2−ΔΔCT) and an internal reference. The primer sequences are presented in Table 1 .
Sequences of primers
| Primers | Sequences |
|---|---|
| VP1 F | 5′-GCT CTA TAG GAG ATA GTG TGA GTA GGG-3′ |
| VP1 R | 5′-ATG ACT GCT CAC CTG CGT GTT-3′ |
| Nrf2 F | 5′-CTC GCT GGA AAA AGA AGT GG-3′ |
| Nrf2 R | 5′-CCG TCC AGG AGT TCA GAG AG-3′ |
| Keap1 F | 5′-TGG CCA GCG TGG AGT GCT AC-3′ |
| Keap1 R | 5′-TTG CAG CAA CAC CCG CTC CA-3′ |
| IL-1β F | 5′-TGA AAT GCC ACC TTT TGA CAG-3′ |
| IL-1β R | 5′-CCA CAG CCA CAA TGA GTG ATA C-3′ |
| IL-6 F | 5′-TGC CTT CTT GGG ACT GAT-3′ |
| IL-6 R | 5′-CTG GCT TTG TCT TTC TTG TT-3′ |
| TNF-α F | 5′-CGA TGA GGT CAA TCT GCC CA-3′ |
| TNF-α R | 5′-CCA GGT CAC TGT CCC AGC-3′ |
| GAPDH F | 5′-GGA AAG CTG TGG CGT GAT-3′ |
| GAPDH R | 5′-AAG GTG GAA GAA TGG GAG TT-3′ |
8
SYBR Green RT-PCR Protocol for Gene Expression
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The cells were subjected to the quantitative RT-PCR with SYBR Green PCR Master Mix (Vazyme, China) and analysed using a Roche RT-PCR System as previously described (Peng et al. 2021 (link)). The primer sequences are given in Additional file 1 : Table S2.
9
Quantitative RT-PCR for Gene Expression Analysis
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The total RNA was extracted using TRIzol solution (Invitrogen). A Primer Script RT Reagent Kit (Takara Bio, Tokyo, Japan) was used to perform reverse transcription of the RNA samples into complement DNA (cDNA). Three independent PCR amplifications were carried out using RT-PCR system (Roche Diagnostics, Basel, Switzerland) with specific primers and iQ SYBR Green Supermix (Bio-Rad Laboratories, Hercules, CA, USA). The PCR cycling was set at 95 °C for 3 min, followed by 39 cycles at 95 °C for 10 s, at 57 °C for 10 s, and at 72 °C for 30 s.
10
Quantifying Gene Expression in MKN-45 Cells
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The mRNA level of DKK1, WNT5A, MDR1, and GAPDH was determined using RT-PCR as previously stated by Mokabber et al. [17] (link) Briefly, total RNA was extracted from the MKN-45 cell line using 1 ml TRIzol (Invitrogen). First-strand complementary DNA (cDNA) was produced from 1 μg total RNA using oligo (dT) primer and M-MLV reverse enzyme (Vivantis, USA). oligo(dT, 1 μl) primer and nuclease-free water were mixed with mRNA, incubated at 65 °C for 5 min and placed on ice for at least 1 min. M-MuLV enzyme (100 u) and buffer (10 × ) were added and incubated at 42 °C for 60 min and then 85 °C for 10 min. Finally, qPCR was done with the SYBR Green PCR Master Mix (EURx, Ltd, Gdañsk, Poland). Real-time PCR reaction was performed in 3 steps: 95 °C for 20 s, followed by 40 cycles of 95 °C for 5 s, and 60 °C for 5 s.
The RT-PCR System (Roche Applied Science) was used to analyze gene expression. Specific human primers DKK1, WNT5A, MDR1, and GAPDH were designed using OLIGO 7.0 software. GAPDH gene was used as a reference gene and relative gene expression was calculated using the CT method [18, 19] . Primer sequences were shown in ▶table 1.
The RT-PCR System (Roche Applied Science) was used to analyze gene expression. Specific human primers DKK1, WNT5A, MDR1, and GAPDH were designed using OLIGO 7.0 software. GAPDH gene was used as a reference gene and relative gene expression was calculated using the CT method [18, 19] . Primer sequences were shown in ▶table 1.
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